Adrienne A. Hilton

Programmed anuclear cell death delimits platelet life span.

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.

Cloning of a murine IL-11 receptor alpha-chain; requirement for gp130 for high affinity binding and signal transduction.

An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha‐chain of the IL‐6 receptor and mRNA was expressed in the 3T3‐L1 pre‐adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor‐dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL‐11 (Kd approximately 10 nM). The capacity to bind IL‐11 with high affinity (Kd = 300‐800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL‐6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL‐11.

Polycomb Repressive Complex 2 (PRC2) Restricts Hematopoietic Stem Cell Activity

Polycomb group proteins are transcriptional repressors that play a central role in the establishment and maintenance of gene expression patterns during development. Using mice with an N-ethyl-N-nitrosourea (ENU)-induced mutation in Suppressor of Zeste 12 (Suz12), a core component of Polycomb Repressive Complex 2 (PRC2), we show here that loss of Suz12 function enhances hematopoietic stem cell (HSC) activity. In addition to these effects on a wild-type genetic background, mutations in Suz12 are sufficient to ameliorate the stem cell defect and thrombocytopenia present in mice that lack the thrombopoietin receptor (c-Mpl). To investigate the molecular targets of the PRC2 complex in the HSC compartment, we examined changes in global patterns of gene expression in cells deficient in Suz12. We identified a distinct set of genes that are regulated by Suz12 in hematopoietic cells, including eight genes that appear to be highly responsive to PRC2 function within this compartment. These data suggest that PRC2 is required to maintain a specific gene expression pattern in hematopoiesis that is indispensable to normal stem cell function.

ERG dependence distinguishes developmental control of hematopoietic stem cell maintenance from hematopoietic specification

Although many genes are known to be critical for early hematopoiesis in the embryo, it remains unclear whether distinct regulatory pathways exist to control hematopoietic specification versus hematopoietic stem cell (HSC) emergence and function. Due to their interaction with key regulators of hematopoietic commitment, particular interest has focused on the role of the ETS family of transcription factors; of these, ERG is predicted to play an important role in the initiation of hematopoiesis, yet we do not know if or when ERG is required. Using in vitro and in vivo models of hematopoiesis and HSC development, we provide strong evidence that ERG is at the center of a distinct regulatory program that is not required for hematopoietic specification or differentiation but is critical for HSC maintenance during embryonic development. We show that, from the fetal period, ERG acts as a direct upstream regulator of Gata2 and Runx1 gene activity. Without ERG, physiological HSC maintenance fails, leading to the rapid exhaustion of definitive hematopoiesis.

Thrombocytopenia and kidney disease in mice with a mutation in the C1galt1 gene

An N-ethyl-N-nitrosourea mutagenesis screen in mice was performed to isolate regulators of circulating platelet number. We report here recessive thrombocytopenia and kidney disease in plt1 mice, which is the result of a severe but partial loss-of-function mutation in the gene encoding glycoprotein-N-acetylgalactosamine-3-β-galactosyltransferase (C1GalT1), an enzyme essential for the synthesis of extended mucin-type O-glycans. Platelet half-life and basic hemostatic parameters were unaffected in plt1/plt1 mice, and the thrombocytopenia and kidney disease were not attenuated on a lymphocyte-deficient rag1-null background. gpIbα and podocalyxin were found to be major underglycosylated proteins in plt1/plt1 platelets and the kidney, respectively, implying that these are key targets for C1GalT1, appropriate glycosylation of which is essential for platelet production and kidney function. Compromised C1GalT1 activity has been associated with immune-mediated diseases in humans, most notably Tn syndrome and IgA nephropathy. The disease in plt1/plt1 mice suggests that, in addition to immune-mediated effects, intrinsic C1Gal-T1 deficiency in megakaryocytes and the kidney may contribute to pathology.

A Novel Mutation in the Nfkb2 Gene Generates an NF-κB2 “Super Repressor”

The noncanonical NF-κB pathway regulates the development and function of multiple organs and cell lineages. We have generated mice harboring a novel mutation in Nfkb2 that prevents the processing of the inhibitory precursor, p100, into the active subunit, p52. Mutant mice express a complex phenotype with abnormalities in a variety of tissues, and with a spectrum that is more severe than in mice carrying a targeted deletion of Nfkb2. Signaling through the noncanonical pathway is ablated due to the absence of p52, resulting in disorganized splenic architecture and disrupted B cell development. The inhibitory precursor form of NF-κB2 interacts with RelA, preventing activation of RelA dimers in response to both canonical and noncanonical stimuli, which in combination with p52 deficiency, results in defective lymph node formation and bone homeostasis. These findings demonstrate a key role for NF-κB2 in the regulation of RelA activation and suggest overlap in the function of NF-κB members in canonical and noncanonical pathway signaling.

Characterization of cDNA and Genomic Clones Encoding Human Myelin Oligodendrocyte Glycoprotein

Abstract: Myelin oligodendrocyte glycoprotein (MOG) is a transmembrane protein expressed only in the CNS and is a possible target autoantigen in multiple sclerosis (MS). To further study the association of MOG with MS, we have characterized cDNA and genomic clones encoding human MOG. The human MOG cDNA, like its rodent and bovine counterparts, encodes a mature protein containing an Ig‐like domain, followed by two potential membrane‐spanning regions. The intron‐exon boundaries of the human MOG gene were mapped and revealed that the signal peptide is encoded by the first exon, the Ig‐like domain of MOG is encoded on the second exon, whereas the remainder of the molecule is encoded by six shorter exons. In addition to the major cDNA species, a second class of MOG cDNA was isolated in which an intron was retained. Not only did this second cDNA species represent 30% of the clones analyzed (nine of 30), but RNA encoding this form was detectable by northern and reverse transcription‐polymerase chain reaction analysis of the brain and spinal cord. Furthermore, we describe several restriction fragment length polymorphisms of the human MOG gene, one of which may be associated with MS susceptibility.

Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl / mice

In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl(-/-) mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl(-/-) p300(Plt6/+) mice displayed elevated platelet counts relative to Mpl(-/-) p300(+/+) controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300(Plt6) mutation, or recipients of Mpl(+/+) p300(Plt6/Plt6) bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl(-/-) thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte development, the inhibition of which allows substantial thrombopoietin (TPO)-independent platelet production.

Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice

With the notable exception of humans, uric acid is degraded to (S)-allantoin in a biochemical pathway catalyzed by urate oxidase, 5-hydroxyisourate (HIU) hydrolase, and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase in most vertebrate species. A point mutation in the gene encoding mouse HIU hydrolase, Urah, that perturbed uric acid metabolism within the liver was discovered during a mutagenesis screen in mice. The predicted substitution of cysteine for tyrosine in a conserved helical region of the mutant-encoded HIU hydrolase resulted in undetectable protein expression. Mice homozygous for this mutation developed elevated platelet counts secondary to excess thrombopoietin production and hepatomegaly. The majority of homozygous mutant mice also developed hepatocellular carcinoma, and tumor development was accelerated by exposure to radiation. The development of hepatomegaly and liver tumors in mice lacking Urah suggests that uric acid metabolites may be toxic and that urate oxidase activity without HIU hydrolase function may affect liver growth and transformation. The absence of HIU hydrolase in humans predicts slowed metabolism of HIU after clinical administration of exogenous urate oxidase in conditions of uric acid-related pathology. The data suggest that prolonged urate oxidase therapy should be combined with careful assessment of toxicity associated with extrahepatic production of uric acid metabolites.

Thrombocytopenia and erythrocytosis in mice with a mutation in the gene encoding the hemoglobin β minor chain

Diverse mutations in the genes encoding hemoglobin (Hb) have been characterized in human disease. We describe here a mutation in the mouse Hbb-b2 gene, denoted Plt12, that precisely mimics the human hemoglobin Hotel Dieu variant. The mutation results in increased affinity of Hb for oxygen and Plt12 mutant mice exhibited reduced partial pressure of O2 in the blood, accompanied by erythrocytosis characterized by elevated erythropoietin levels and splenomegaly with excess erythropoiesis. Most homozygous Hbb-b2Plt12/Plt12 mice succumbed to early lethality associated with emphysema, cardiac abnormalities, and liver degeneration. Survivors displayed a marked thrombocytopenia without significant deficiencies in the numbers of megakaryocytes or megakaryocyte progenitor cells. The lifespan of platelets in the circulation of Hbb-b2Plt12/Plt12 mice was normal, and splenectomy did not correct the thrombocytopenia, suggesting that increased sequestration was unlikely to be a major contributor. These data, together with the observation that megakaryocytes in Hbb-b2Plt12/Plt12 mice appeared smaller and deficient in cytoplasm, support a model in which hypoxia causes thrombocytopenia as a consequence of an inability of megakaryocytes, once formed, to properly mature and produce sufficient platelets. The Plt12 mouse is a model of high O2-affinity hemoglobinopathy and provides insights into hematopoiesis under conditions of chronic hypoxia.