Background-Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1) controls the intracellular level of the phosphoinositide 3-kinase product phosphotidylinositol-3,4,5-trisphosphate and functions as a negative regulator of cytokine and immune receptor signaling. Emerging evidence suggests that the phosphoinositide 3-kinase pathway might be involved in allergic inflammation in the lung. However, the functional relevance of SHIP-1 in the T H 2 activation pathway has not been established. SHIP-1 −/− mice have spontaneous myeloproliferative inflammation in the lung, the nature of which has not been elucidated. We hypothesized that SHIP-1 plays an important role as a regulator in pulmonary allergic inflammation and in maintaining lung homeostasis.

No abstract

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.

The activation of Fli-1, an Ets transcription factor, is the critical genetic event in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. Fli-1 overexpression leads to erythropoietin-dependent erythroblast proliferation, enhanced survival, and inhibition of terminal differentiation, through activation of the Ras pathway. However, the mechanism by which Fli-1 activates this signal transduction pathway has yet to be identified. Down-regulation of the Src homology 2 (SH2) domain-containing inositol-5-phosphatase-1 (SHIP-1) is associated with erythropoietin-stimulated IntroductionThe progression of cancer is a multistep process in which oncogenes and tumor suppressor genes mediate changes in gene expression required for malignant transformation. Transcription factors, often described as oncogenes or tumor suppressor genes, play a pivotal role within signal transduction pathways governing cellular proliferation, differentiation, and apoptosis. 1 In Friend murine leukemia virus (F-MuLV)-induced erythroleukemia, the transcription factor fli-1 is activated as a result of proviral integration. A proto-oncogene and member of the Ets family of transcription factors, fli-1 plays a critical role in normal development, hematopoiesis and oncogenesis. [2][3][4][5][6] Accordingly, a recent study from our group, and that of another laboratory, has demonstrated that Fli-1 inhibition suppresses growth and induces cell death in murine and human erythroleukemias. 7,8 Fli-1 overexpression in erythroblasts blocks erythroid differentiation that is associated with activation of the Shc/Ras pathway in response to erythropoietin (Epo) stimulation. 9 The Src homology 2 (SH2) domain-containing inositol-5-phosphatase-1 (SHIP-1) is associated with phosphorylated Shc in Epo-stimulated erythroleukemic cells, 9 and correlates with increased proliferation of transformed erythroid cells. 10 Because SHIP-1 is involved in the regulation of extracellular signal-regulated kinase/mitogenactivated protein kinase (ERK/MAPK) and phosphatidylinositol 3-kinase (PI 3-K) signaling pathways, 11,12 we hypothesized that Fli-1 may promote Epo-induced proliferation, and activation of the above mentioned pathways, in part, through regulation of SHIP-1.SHIP-1 plays a role in the activation and proliferation of myeloid cells, macrophages, and mast cells. This phosphatase also negatively regulates c-Jun NH2-terminal kinase (JNK), 13 and nuclear factor-B (NF-B) activity. 14 SHIP-1 is expressed exclusively in hematopoietic cells and developing spermatogonia and is activated after cytokine, growth factor, or immunoreceptor activation in hematopoietic cells. 12,14,15 SHIP-1 is expressed in mature T cells, granulocytes, monocytes/macrophages, mast cells, and platelets. 16 Interestingly, it has been documented that SHIP-1 expression is turned off during erythropoiesis, specifically in TER119ϩ erythroid cells. 15 Expression of a short form of SHIP-1 (termed s-SHIP) modulates the activation threshold of primitive stem cells. 17,18 SHIP-1 knockout mice are viable b...

The ability of random mutagenesis techniques to annotate the mammalian genome can be hampered due to genetic redundancy and compensatory pathways that mask heterozygous mutations under homeostatic conditions. The objective of this study was to devise a pharmacologically sensitized screen using the chemotherapeutic drug, 5-fluorouracil (5FU), to induce cytopenia. 5FU dose was optimized in the 129/SvImJ, C57BL/6J, BALB/cJ, and C3H/HeJ strains of laboratory mice. N-ethyl-N-nitrosourea (ENU) mutagenesis was performed on 129/SvImJ males and phenotypic variants were identified by backcrossing on to the C57BL/6J background. G1 animals were challenged with 100 μg/g 5FU and phenodeviants with altered platelet recovery were monitored. Of 546 G1 animals tested, 15 phenodeviants were identified that displayed increased baseline platelet number, a platelet overshoot, or delayed platelet recovery, thereby demonstrating the utility of this approach for uncovering mutations in megakaryocyte and platelet development. Four G1 mice were selected for further analysis. The phenotypes were heritable in all four strains and genetic mapping identified a chromosome location in two of the three G2 lines tested. In conclusion, our group has developed a sensitized random mutagenesis screen utilizing 5FU and has shown that the strain combination of 129/SvImJ × C57BL/6J is robust for identification of founder lines with defects in megakaryocyte and platelet development.

BCR-ABL induces chronic myeloid leukemia (CML) through the aberrant regulation of multiple signaling substrates. Previous research has shown that BCR-ABL mediates down-modulation of CBL-B protein levels. A murine bone marrow transplantation (BMT) study was performed to assess the contribution of Cbl-b to BCR-ABL-induced disease. The predominant phenotype in the Cbl-b(-/-) recipients was a CML-like myeloproliferative disease (MPD) similar to that observed in the wild-type animals, but with a longer latency, diminished circulating leukocyte numbers and reduced spleen weights. Despite the decreased leukemic burden in comparison to their wild-type counterparts, the Cbl-b(-/-) animals displayed enhanced numbers of Gr-1(+)/Mac-1(+) spleen cells and neutrophilia. On the basis of prior evidence of CBL-B-dependent motility toward SDF-1α, we hypothesized that Cbl-b deficiency might impair bone marrow localization during transplantation. Homing experiments showed reduced migration of Cbl-b(-/-) cells to the bone marrow. Intrafemoral transplantation of BCR-ABL-transduced Cbl-b(-/-) cells revealed equivalent latency of disease development to the wild-type transplants, supporting the conclusion that Cbl-b deficiency diminishes homing of leukemic cells to the bone marrow, and perturbs the proliferation of BCR-ABL-expressing malignant clones during CML development.

2527 May-Hegglin Anomaly was originally defined as patients with macrothrombocytopenia. It is now recognized that the phenotypes of May-Hegglin Anomaly and the related syndromes described by Epstein, Fechtner and Sebastian overlap comprising macrothrombocytopenia, neutrophil inclusions, deafness, cataract formation and nephritis. These disorders are defined by mutations in the non-muscle myosin heavy chain IIA gene, MYH9 and have been termed MYH9-Related Disorders. Myh9 is required during murine embryogenesis as Myh9-null embryos die of patterning defects. Megakaryocyte-specific deletion of Myh9 has been performed by the Gachet group and Shivdasani and colleagues examined megakaryocyte function in Myh9-deficient embryonic stem cells induced to differentiate in vitro. The phenotypes of these mice include increased bleeding times, absence of clot retraction and defective Integrin β3 phosphorylation coupling to impaired activation of downstream Rho-Rock signalling and lamellipodia formation. To date, no knock-in MYH9 alleles corresponding to MYH9-related Disorder mutations have been reported. MYH9-related Disorders frequently are misdiagnosed as Chronic Immune Thrombocytopenia, so having enhanced knowledge of the molecular etiology of mutant MYH9 alleles is a priority. Our group has been interested in utilizing random mutagenesis to identify novel alleles of human hematopoietic disorders. We performed a dominant screen in which 129 male mice were mutagenized with ENU and back-crossing was performed on the C57BL/6 background. We identified a heritable line (7238) with macrothrombocytopenia that mapped to a 4.25 Mb region on mouse Chromosome 15. Due to our inability to further refine the interval through back-crossing, we performed high throughput sequencing by comparing SNPs between 129, C57BL/6, 7238/+ and 7238/7238 mice. We identified 18 novel variations, including 1 that corresponded to a coding change - Myh9 Q1443L. Subsequent Sanger sequencing and back-crossing eliminated the remaining 17 variations, confirming our assignment of the 7238 phenotype to a mutation of Myh9. The Myh9 Q1443L allele is within proximity of the MYH9 D1424Y/N/H mutations found within clinical specimens. Myh9 Q1443L/Q1443L mice are viable allowing us to examine the effect of mutation of one or both alleles of Myh9. Myh9Q1443L/Q1443L mice had increased bleeding times, whereas Myh9Q1443L/+ mice were identical to wild type mice. Platelet aggregation experiments were performed after ADP or thrombin stimulation. Decreased aggregation was observed in a dosage-dependent manner, with Myh9Q1443L/+ mice displaying intermediate levels when compared with Myh9Q1443L/Q1443L co-hort. Adherence and aggregation of Myh9Q1443L/Q1443L platelets to a collagen matrix was decreased at shear rates of 350/s and 1800/s. Staining of Myh9Q1443L/+ and Myh9Q1443L/Q1443L neutrophils also revealed neutrophil inclusions. In addition to the hematopoietic phenotypes, we have observed increased cataract formation with a frequency of 36% in wild type animals, 60% in Myh9Q1443L/+ and 92% in Myh9Q1443L/Q1443L mice. Sporadic hematuria and proteinuria is found in Myh9Q1443L mice. We are currently performing albumen loading and lipopolysaccharide activation experiments to determine whether 7238 mice have altered response to nephric stress. We are crossing 7238 mice to the DBA genetic background, which is more sensitive to subtle kidney phenotypes. We performed auditory brain response measurements on aged wild type, Myh9Q1443L/+ and Myh9Q1443L/Q1443L mice and observed no differences in frequency responses ranging from 2 to 24kHz. Myh9Q1443L/+ mice displayed hypertrophy of the preputial gland whereas Myh9Q1443L/Q1443L males showed atrophy of this gland and Myh9Q1443L/Q1443L male mice have reduced fertility. No differences in fertility have been described to date in MYH9 related disorders. In conclusion, Myh9Q1443L/Q1443L mice display macrothrombocytopenia, neutrophil inclusions and cataract formation described in MYH9-related disorders and represent the first animal model of May-Hegglin Anomaly. Disclosures: No relevant conflicts of interest to declare.

Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2 K915X), resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2 +/-). Timed pregnancy experiments revealed that Jak2 K915X/K915X and Jak2 -/- displayed embryonic lethality; however, Jak2 K915X/K915X embryos were viable an additional two days compared to Jak2 -/- embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.