GATA1-deficient Mice Research Articles

GATA factor-related hematopoietic diseases

GATA1-deficient mice die in utero on 12.5 embryonic day (E12.5) due to a complete block of primitive erythropoiesis in the yolk sac, while GATA2-deficient mice die on E10.5 due to severe anemia and hemorrhage, since GATA2 is essential for the development of hemangioblasts, which are common precursor cells of hematopoietic stem cells and endothelial cells. In contrast, GATA3 is critical to the development of Th2 cells. However, GATA3-deficient mice die in utero before the particular phenotype of hematopoietic system emerges, which is caused by a defect in the development of nervous and renal urinary systems. It has been well elucidated that defects in the hematopoietic GATA factors disturb hematopoietic homeostasis. However, details on how GATA factor dysfunction leads to human hematopoietic diseases remain to be clarified. At the end of the twentieth century, several mutations in GATA1 gene were identified as the cause of familial thrombocytopenia. Since then, various types of hematopoietic diseases elicited by GATA1 and GATA2 dysfunctions have been reported. This review summarizes recent topics of GATA factor-related hematopoietic diseases.

Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development.

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

The transcription factor GATA1 regulates NBEAL2 expression through a long-distance enhancer.

Gray platelet syndrome is named after the gray appearance of platelets due to the absence of α-granules. It is caused by recessive mutations in NBEAL2, resulting in macrothrombocytopenia and myelofibrosis. Though using the term gray platelets for GATA1 deficiency has been debated, a reduced number of α-granules has been described for macrothrombocytopenia due to GATA1 mutations. We compared platelet size and number of α-granules for two NBEAL2 and two GATA1-deficient patients and found reduced numbers of α-granules for all, with the defect being more pronounced for NBEAL2 deficiency. We further hypothesized that the granule defect for GATA1 is due to a defective control of NBEAL2 expression. Remarkably, platelets from two patients, and Gata1-deficient mice, expressed almost no NBEAL2. The differentiation of GATA1 patient-derived CD34+ stem cells to megakaryocytes showed defective proplatelet and α-granule formation with strongly reduced NBEAL2 protein and ribonucleic acid expression. Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of NBEAL2 covered by a H3K4Me1 mark indicative of an enhancer locus. Luciferase reporter constructs containing this region confirmed its enhancer activity in K562 cells, and mutagenesis of the GATA1 binding sites resulted in significantly reduced enhancer activity. Moreover, DNA binding studies showed that GATA1 and GATA2 physically interact with this enhancer region. GATA1 depletion using small interfering ribonucleic acid in K562 cells also resulted in reduced NBEAL2 expression. In conclusion, we herein show a long-distance regulatory region with GATA1 binding sites as being a strong enhancer for NBEAL2 expression.

GATA-related hematologic disorders.

The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice invivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.

GATA1 Changes DNA-Binding Fashion in a Binding-Site-Specific Manner and Alters Transcriptional Activity during Erythropoiesis

GATA1 is a transcription factor that coordinately regulates multiple target genes during the development and differentiation of erythroid and megakaryocytic lineages through binding to GATA motif (A/T)GATA(A/G). GATA1 has four functional domains, i.e., two transactivation domains reside in amino- and carboxyl- terminus, which transactivate GATA1 target genes redundantly and/or cooperatively, and two zinc-finger domains in the middle of the protein. The two zinc finger domains of GATA1 have been characterized extensively and their links to human diseases have also been identified. Carboxyl-terminal side zinc (C)-finger is essential for the DNA binding of GATA1, whereas amino-terminal side zinc (N)-finger retains insufficient binding activity to the GATA motifs by itself, but contributes to stabilize the binding of C-finger to a double GATA site arranged in a palindromic manner. Of note, while this two-finger structure is conserved in six distinct vertebrate GATA factors, there exist GATA factors with single zinc finger in non-vertebrates, indicating that only the C-finger and following basic tail region are evolutionary conserved in both vertebrate and non-vertebrate GATA factors. In our transgenic rescue analyses, GATA1 lacking the N-finger (ΔNF-GATA1) supports, if not completely, the erythropoiesis in mice, but mice without C-finger (ΔCF-GATA1) die in utero showing similar phenotype to the mice with complete loss-of-GATA1-function. Therefore, roles that the N-finger plays have been assumed to be evolutionally acquired features during molecular evolution.In this study, we have examined GATA-motif configuration-specific modulation of GATA1 function by using composite GATA elements in which two GATA motifs aligned side-by-side, either tandem or palindromic. We have defined changes in the GATA1 binding and transactivation activity in accordance with the arrangement of cis -acting GATA motifs. While GATA1 binds to Single-GATA in a monovalent way via C-finger without the influence of N-finger, the N-finger appears to contribute to specific bivalent binding of GATA1 to Pal-GATA, i.e., the N- and C-fingers in a single GATA1 molecule individually bind to two GATA motifs aligned in a palindromic orientation. Showing very good agreement with the human case analyses, the transgenic expression of G1R216Q that lacks N-finger-DNA interaction potential hardly rescues the GATA1-deficient mice due to defects in definitive erythropoiesis, indicating that roles owed by R216 residue are vital for the GATA1 activity in vivo. The N-finger also contributes to GATA1 homodimer formation, which is a prerequisite for two GATA1 binding to two GATA motifs aligned in a tandem orientation. Each GATA1 C-finger in the dimeric GATA1 protein binds to each GATA motif in Tandem-GATA. In this regard, we previously found in a transgenic complementation rescue assay that mutant GATA1 molecule G13KA, which lacks the dimerization potential but possesses most of the other N- and C-finger functions, hardly rescues the GATA1-deficient mice from embryonic lethality, indicating that the GATA1 dimerization is important to attain full GATA1 activity. We surmise based on these observations that the configuration of cis -acting GATA motifs located in the regulatory regions of the GATA1 target genes critically influences the DNA-binding of GATA1 and controls transcription of the genes. DisclosuresNo relevant conflicts of interest to declare.

The effects of GATA-1 and NF-E2 deficiency on bone biomechanical, biochemical, and mineral properties.

Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone.

DNA Binding Diversity Achieved Through the Interaction of GATA1 N-Finger and GATA Motif Is Important for Embryonic Erythropoiesis

AbstractAbstract 3441Transcription factor GATA1 regulates a set of genes essential for the erythroid and megakaryocytic cell differentiation through the interaction with GATA motifs (consensus sequence: A/TGATAA/T). Two zinc fingers within GATA1 have been identified to be important in the DNA binding of GATA1, which are referred to as C-finger (CF) and N-finger (NF) domains. It has been shown that transactivation activity of GATA1 is completely abolished upon deletion of the CF domain, indicating that the CF domain is a requisite for the DNA binding of GATA1. While conventional reporter transactivation analyses hardly clarified the importance of the NF domain for the DNA binding, substitution mutations on 216th arginine (R216) located in the DNA-interacting surface of the NF domain have been identified to cause familial diseases of thrombocytopenia, thalassemia, and porphyria. As a consequence of the substitution of R216 to glutamine (Q) or tryptophan (W), DNA binding activity of GATA1 to a palindromic configuration of two GATA motifs (palindromic GATA) was largely diminished, while that to a single GATA motif was maintained.In this study we have examined the DNA binding diversity of GATA1 caused by the difference in the configuration of GATA motifs. We performed surface plasmon resonance (SPR) analyses of GATA1 to a single GATA, a palindromic GATA, and a repeating configuration of two GATA motifs (tandem GATA). We found that GATA1 binds to the palindromic GATA motif in a bivalent way, while it binds to the single GATA motif in a monovalent mode. We also found that a double quantity of GATA1 is associated with the tandem GATA motif and GATA1 lacking the NF domain binds to any configurations of GATA motif in a monovalent way.To further investigate contribution of the NF domain to the binding mode of GATA1, we have constructed two types of GATA1 mutants; one type was the substitution mutations on R216 (R216Q and R216W) that were mouse homologues of the human mutations, while the other type was the alanine substitution mutation on three lysine residues (K245, K246 and K312; referred to as 3KA mutant), whereby dimerization potential of GATA1 was reduced to trace level similar to the case for GATA1 lacking the NF domain. Impotantly, R216Q and R216W mutants bind the palindromic GATA motif in a monovalent way, while these mutants bind normally to the other configuration of GATA motifs. In contrast, we found that one molecule of 3KA mutant bound to the tandem GATA motif and this observation seems to explain well the fact that dimerization potential of GATA1 is an important requisite for the full-function of GATA1 in embryos. The binding modes of this 3KA mutant to the other configurations were not influenced.These results thus demonstrate that the both NF and CF domains recognize the multiple configurations of GATA motifs and specify the binding modes of GATA1. Importantly, GATA1-deficient mice rescued with R216Q were lethal during late gestation period due to abnormality in erythroid differentiation, indicating that the contribution of the NF domain to the recognition of the palindromic GATA motif configuration indeed functions in vivo. These results thus support our contention that the NF domain acts to regulate a proper spatio-temporal gene expression of a subset of GATA1 target genes utilizing the variations in the GATA motif configuration. Disclosures:No relevant conflicts of interest to declare.

N- and C-terminal Transactivation Domains of GATA1 Protein Coordinate Hematopoietic Program

Transcription factor GATA1 regulates the expression of a cluster of genes important for hematopoietic cell differentiation toward erythroid and megakaryocytic lineages. Three functional domains have been identified in GATA1, a transactivation domain located in the N terminus (N-TAD) and two zinc finger domains located in the middle of the molecule. Although N-TAD is known as a solitary transactivation domain for GATA1, clinical observations in Down syndrome leukemia suggest that there may be additional transactivation domains. In this study, we found in reporter co-transfection assays that transactivation activity of GATA1 was markedly reduced by deletion of the C-terminal 95 amino acids without significant attenuation of the DNA binding activity or self-association potential. We therefore generated transgenic mouse lines that expressed GATA1 lacking the C-terminal region (GATA1-ΔCT). When we crossed these transgenic mouse lines to the Gata1-deficient mouse, we found that the GATA1-ΔCT transgene rescued Gata1-deficient mice from embryonic lethality. The embryos rescued with an almost similar level of GATA1-ΔCT to endogenous GATA1 developed beyond embryonic 13.5 days, showing severe anemia with accumulation of immature erythroid cells, as was the case for the embryos rescued by endogenous levels of GATA1 lacking N-TAD (GATA1-ΔNT). Distinct sets of target genes were affected in the embryos rescued by GATA1-ΔCT and GATA1-ΔNT. We also found attenuated GATA1 function in cell cycle control of immature megakaryocytes in both lines of rescued embryos. These results thus demonstrate that GATA1 has two independent transactivation domains, N-TAD and C-TAD. Both N-TAD and C-TAD retain redundant as well as specific activities for proper hematopoiesis in vivo.

Eosinophil-mediated lung host defense against Pneumocystis murina (118.6)

Abstract We have recently reported that Src TKO mice, which demonstrate augmented lung clearance of P. murina (PM) (Infect Immun, May 2009), have enhanced expression of alternative macrophage activation (M2a) markers and the M2a-associated chemokines CCL17 and CCL22 (J Immunol, February 2011). As CCL17 and CCL22 often drive the recruitment of eosinophils, we questioned their role in PM lung defense. PM infection resulted in significantly increased expression of eosinophil peroxidase (Epx) mRNA, which was further enhanced in mice with greater resistance to PM lung infection. We further observed PM-induced mRNA expression of Prg2, but not Ear1 or Ear2, in the lungs. GATA1 deficient mice, which have a selective deficiency in eosinophils, demonstrated impaired PM lung clearance. In turn, Epx and Prg2 mRNA expression was significantly lower in GATA1 deficient mice. Finally, bone marrow-derived eosinophils exhibited the capacity to kill PM in vitro. Collectively, our work supports a role for eosinophils in lung host defense against Pneumocystis.

Mature erythrocyte membrane homeostasis is compromised by loss of the GATA1-FOG1 interaction

AbstractGATA1 plays essential roles in erythroid gene expression. The N-terminal finger of GATA1 (GATA1-Nf) is important for association with FOG1. Substitution mutations in GATA1-Nf, such as GATA1V205M that diminish the GATA1-FOG1 association, have been identified in human thrombocytopenia and anemia cases. A mouse model of human thrombocytopenia has been established using a transgenic complementation rescue approach; GATA1-deficient mice were successfully rescued from embryonic lethality by excess expression of GATA1V205G, but rescued adult mice suffered from severe thrombocytopenia. In this study, we examined GATA1-deficient mice rescued with GATA1V205G at a comparable level to endogenous GATA1. Mice rescued with this level of GATA1V205G rarely survive to adulthood. Rescued newborns suffered from severe anemia and jaundice accompanied with anisocytosis and spherocytosis. Expression of Slc4a1, Spna1, and Aqp1 genes (encoding the membrane proteins band-3, α-spectrin, and aquaporin-1, respectively) were strikingly diminished, whereas expression of other canonical GATA1-target genes, such as Alas2, were little affected. Lack of these membrane proteins provoked perturbation of membrane skeleton. Importantly, the red cells exhibited increased reactive oxygen species accumulation. These results thus demonstrate that the loss of the GATA1-FOG1 interaction causes a unique combination of membrane protein deficiency and disturbs the function of GATA1 in maintaining erythroid homeostasis.

Two Distinct Transactivation Domains of GATA1 Contribute Megakaryopoiesis,

Abstract 3384Transcription factor GATA1 orchestrates erythroid/megakaryocytic cell differentiation by coordinately regulating the expression of multiple genes encoding proteins responsible for the proliferation, differentiation and survival of progenitors. Mice lacking GATA1 expression die in utero due to insufficient primitive erythropoiesis. GATA1 retains three functional domains; N-terminus (NT) transactivation domain (TAD) and N-terminus and C-terminus zinc finger domains that are both critical for DNA-binding and interaction with co-factors. We previously showed that each domain of GATA1 confers distinguishable properties to GATA1 during primitive and definitive hematopoiesis in vivo. Especially, GATA1 mutant lacking NT domain (GATA1s) is sufficient to rescue the GATA1-deficient mice from embryonic lethality, but the rescued mice suffered from anemia and thrombocytopenia. Showing very good agreement with this finding, inherited mutation yielding GATA1s was found in a family with dyserythropoiesis and dysmegakaryopoiesis. Whereas previous biochemical studies argued that NT domain was a unique TAD in GATA1, these findings suggest that alternative TAD domain is vital for the GATA1 activity in vivo. In this study, we focus on the C-terminus (CT) region of GATA1. The transactivation activity of GATA1 lacking the CT region (G1-DCT) was significantly reduced to approximately 30% of the wild-type GATA1, which was similar to the GATA1s or NT deletion level. Practically no transactivation activity was found in the GATA1 lacking both NT and CT domains. We also examined transactivation potential of the NT and CT fused with GAL4-DBD. CT-GAL4-DBD fusion protein showed Luciferase reporter activity over 20-fold higher than the GAL4-DBD. Furthermore, retrovirally transduced G1-DCT failed to control the hyperproliferation of megakaryocyte progenitors lacking endogenous GATA1, similar to the salient feature reported for GATA1s. These results thus indicate that the coordinated function of CT- and NT-TADs of GATA1 is required in the megakaryopoiesis. GATA1s mutation was found in majority cases of Down syndrome patients with transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Since CT domain is rich in Ser, Thr and Pro residues, while the NT domain is an acidic transactivation domain rich in Glu, we surmise that GATA1 regulates target gene expression by utilizing two distinct transactivation domains. The imbalance in gene expression in megakaryocytic progenitors, which is caused by GATA1s loosing the function of NT-TAD, may be the key aspect for the understanding of the onset of TMD and AMKL. Disclosures:No relevant conflicts of interest to declare.

Immature and mature megakaryocytes enhance osteoblast proliferation and inhibit osteoclast formation

Recent data suggest that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA-1 deficient mice. GATA-1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA-1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA-1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC formation that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA-1 deficient MKs survive longer than wild-type controls. So while increased MK number in GATA-1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA-1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs.

Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of a GATA1 Protein Lacking the N-terminal Domain

GATA1 is essential for the differentiation of erythroid cells and megakaryocytes. The Gata1 gene is composed of multiple untranslated first exons and five common coding exons. The erythroid first exon (IE exon) is important for Gata1 gene expression in hematopoietic lineages. Because previous IE exon knockdown analyses resulted in embryonic lethality, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the floxed IE exon in adulthood using an inducible Cre expression system. In this conditional knock-out mouse line, the Gata1 mRNA level was significantly down-regulated in the megakaryocyte lineage, resulting in thrombocytopenia with a marked proliferation of megakaryocytes. By contrast, in the erythroid lineage, Gata1 mRNA was expressed abundantly utilizing alternative first exons. Especially, the IEb/c and newly identified IEd exons were transcribed at a level comparable with that of the IE exon in control mice. Surprisingly, in the IE-null mouse, these transcripts failed to produce full-length GATA1 protein, but instead yielded GATA1 lacking the N-terminal domain inefficiently. With low level expression of the short form of GATA1, IE-null mice showed severe anemia with skewed erythroid maturation. Notably, the hematological phenotypes of adult IE-null mice substantially differ from those observed in mice harboring conditional ablation of the entire Gata1 gene. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and selecting the correct transcription start site of the Gata1 gene.

Spherocytic Hemolytic Anemia Caused by Disruption of GATA1-FOG1 Interaction.

Abstract 775Transcription factor GATA1 is essential for erythroid/megakaryocytic cell differentiation. Gene ablation studies have revealed that GATA1 plays essential roles in the differentiation of erythroid and megakaryocytic lineages. Two functional zinc finger domains have been identified within the GATA1 molecule, and N-terminal finger (NF) is required for the association with cofactor FOG1 and also DNA recognition. Several substitution mutations in the GATA1 NF that diminish association of GATA1-FOG1 have been identified in patients with thrombocytopenia and anemia. We previously established a genetically engineered mouse model, which mimics a human disease X-linked thrombocytopenia with inherited GATA1 mutation, utilizing the transgenic complementation rescue approach. GATA1-deficient mice were successfully rescued from embryonic lethality by excess expression of mutant GATA1 (GATA1V205G) and showed severe thrombocytopenia with impaired cytoplasmic maturation of megakaryocytes resembling the human disease. However, these mice did not show dyserythropoietic phenotype except for the attenuated erythropoiesis under stress conditions. On the contrary, GATA1-deficient mice rescued by transgenic expression of GATA1V205G at the level comparable with that of the endogenous GATA1 hardly survived though the postnatal stage. Accordingly, we analyzed rescued embryos during perinatal period. At 18.5 embryonic days, expected number of rescued embryos survived although they showed macroscopic anemia, indicating that embryonic erythropoiesis was not fully sustained by transgenic expression of GATA1V205G at the endogenous level. Surprisingly, rescued newborns suffered from severe anemia and jaundice. Serum indirect bilirubin level was significantly elevated and circulating erythrocytes showed abnormal morphology characterized as spherocytes by means of scanning electron microscopic observation. The expressions of Band3 gene/protein were strikingly diminished in the rescued erythroid cells, while expression of the other membrane related genes varied in individuals. Thus, these results indicate that hemolytic event associated with the lack of Band3 and dysfunction of erythrocyte membrane is induced during perinatal period, and GATA1 maintains the erythroid membrane homeostasis through interaction with FOG1. Disclosures:No relevant conflicts of interest to declare.

PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications

Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34(+) cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34(+) cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Inhibition of VPAC1 Signaling Raises Platelet Numbers after Myelosuppressive Therapy and in GATA1 Congenital Thrombocytopenia.

Thrombocytopenia is a serious complication in patients undergoing myelosuppressive chemotherapy. Therefore, the development of new strategies to reduce thrombocytopenia or accelerate platelet recovery without adverse effect on the bone marrow (BM) is still warranted. Previously we found that the pituitary adenylate cyclase activating peptide (PACAP) is a physiological inhibitor of platelet activation (JCI, 2004). The receptor for PACAP on megakaryocytes (MKs) and platelets is the Gs coupled receptor VPAC1. We have presently investigated whether the neutralization of the regulatory role of VPAC1 can affect thrombopoiesis, independently of thrombopoietin (TPO). We found that inhibition of VPAC1 signaling stimulates in vitro megakaryopoiesis, since more MKs could be generated from Sca1+ cells, isolated from normal mouse BM by incubation with a neutralizing anti-VPAC1 antibody (Ab) (23A11). 23A11 was also capable of enhancing the later stages of MK proliferation of human cord blood derived CD34+ cells. These in vitro findings were validated in two in vivo animal models of myelosuppressive therapy. The subcutaneous injection of a neutralizing Ab directed against PACAP (PP1A4) or 23A11 (1 mg/kg on days 0,3 and 7) boosted platelet recovery in mice after chemotherapy-induced thrombocytopenia (20 mg/kg busulfan intraperitoneally on days 7 and 10) in a TPO-independent manner. Platelet recovery in mice, injected with PP1A4, was also significantly increased compared to non-treated mice exposed to 8 Gy total body irradiation. Plasma TPO concentrations did not differ between the two groups. We next questioned whether VPAC1 inhibition could rescue the GATA1-defective phenotype due to a developmental arrest in the megakaryoblast stage. FACS analysis of BM Sca1+ cells from GATA1 deficient mice cultured during 12 days in MK differentation medium in vitro showed an increased MK DNA ploidy when 23A11 was added to the culture. A similar observation was made with CD34+ cells from BM of a GATA1 deficient patient with severe thrombocytopenia. GATA1 deficient mice are thrombocytopenic, with a platelet count of 50 +/− 15 x 10(9) plt/L. This platelet number increases to 150 +/− 40 x 10(9) plt/L after subcutaneous injection with 23A11 whereas injection with PBS had no effect. Again, TPO plasma levels in these injected GATA1 deficient mice showed no association between anti-PACAP or anti-VPAC1 treatment. Thus, our data indicate that VPAC1 signaling tempers normal megakaryopoiesis and that elimination of this pathway facilitates platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages.Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

Selective impairment of platelet activation to collagen in the absence of GATA1

AbstractDefects in the X-linked DNA-binding megakaryocyte transcription factor GATA1 cause thrombocytopenia and abnormal platelet function. However, detailed studies of GATA1 function in platelet activation are lacking. Here, we studied platelets from GATA1-deficient mice and from a male patient (S14) with a bleeding diathesis attributed to a single amino acid substitution (R216Q) in the N-terminal GATA1 zinc finger that alters binding to DNA. In both cases there was inhibition of aggregation to collagen and decreased tyrosine phosphorylation of glycoprotein VI (GPVI)–signaling proteins. This effect was more marked in GATA1-deficient murine platelets, where it was associated with a significant reduction in surface GPVI expression. Moreover, both human and murine GATA1-mutant platelets showed reduced adhesion and aggregate formation on a collagen matrix at an intermediate rate of shear, although this could not be accounted solely by the thrombocytopenia and altered GPVI expression, indicating that GATA1 regulates additional factors important for platelet activation under shear. In contrast, there was no inhibition of responses to G protein–coupled receptor agonists in GATA1-perturbed platelets. Our results are consistent with GATA1 regulating some but not all pathways of platelet activation, leading to an impairment of aggregate formation under flow, which cannot be attributed solely to the thrombocytopenia.

Transgenic rescue of GATA-1–deficient mice with GATA-1 lacking a FOG-1 association site phenocopies patients with X-linked thrombocytopenia

AbstractAssociation of GATA-1 and its cofactor Friend of GATA-1 (FOG-1) is essential for erythroid and megakaryocyte development. To assess functions of GATA-1–FOG-1 association during mouse development, we used theGATA-1hematopoietic regulatory domain to generate transgenic mouse lines expressing a mutant GATA-1, which contains a substitution of glycine 205 for valine (V205G) that abrogates its association with FOG-1. We examined whether the transgenic expression of mutant GATA-1 rescuesGATA-1germ line mutants from embryonic lethality. In high-expressor lines we observed that the GATA-1V205Grescues GATA-1–deficient mice from embryonic lethality at the expected frequency, revealing that excess GATA-1V205Gcan eliminate the lethal anemia that is due to GATA-1 deficiency. In contrast, transgene expression comparable to the endogenous GATA-1 level resulted in much lower frequency of rescue, indicating that the GATA-1–FOG-1 association is critical for normal embryonic hematopoiesis. Rescued mice in these analyses exhibit thrombocytopenia and display dysregulated proliferation and impaired cytoplasmic maturation of megakaryocytes. Although anemia is not observed under steady-state conditions, stress erythropoiesis is attenuated in the rescued mice. Our findings reveal an indispensable role for the association of GATA-1 and FOG-1 during late-stage megakaryopoiesis and provide a unique model for X-linked thrombocytopenia with inheritedGATA-1mutation.

Essential and instructive roles of GATA factors in eosinophil development.

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1–deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.