Negative regulation of hematopoiesis by the fused in myeloproliferative disorders gene product

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.     

hnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta-tropomyosin exon 6B

Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them.     

Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I

Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.     

Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

We have recently isolated a novel cytomatrix at the active zone (CAZ)-associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.     

CXCR2 is critical to hyperoxia-induced lung injury

Hyperoxia-induced lung injury is characterized by infiltration of activated neutrophils in conjunction with endothelial and epithelial cell injury, followed by fibrogenesis. Specific mechanisms recruiting neutrophils to the lung during hyperoxia-induced lung injury have not been fully elucidated. Because CXCL1 and CXCL2/3, acting through CXCR2, are potent neutrophil chemoattractants, we investigated their role in mediating hyperoxia-induced lung injury. Under variable concentrations of oxygen, murine survival during hyperoxia-induced lung injury was dose dependent. Eighty percent oxygen was associated with 50% mortality at 6 days, while greater oxygen concentrations were more lethal. Using 80% oxygen, we found that lungs harvested at day 6 demonstrated markedly increased neutrophil sequestration and lung injury. Expression of CXCR2 ligands paralleled neutrophil recruitment to the lung and CXCR2 mRNA expression. Inhibition of CXC chemokine ligands/CXCR2 interaction using CXCR2(-/-) mice exposed to hyperoxia significantly reduced neutrophil sequestration and lung injury, and led to a significant survival advantage as compared with CXCR2(+/+) mice. These findings demonstrate that CXC chemokine ligand/CXCR2 biological axis is critical during the pathogenesis of hyperoxia-induced lung injury.     

Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.     

Depletion of CXCR2 inhibits tumor growth and angiogenesis in a murine model of lung cancer

The Glu-Leu-Arg(+) (ELR(+)) CXC chemokines are potent promoters of angiogenesis and have been demonstrated to induce a significant portion of nonsmall cell lung cancer-derived angiogenic activity and support tumorigenesis. ELR(+) CXC chemokines share a common chemokine receptor, CXCR2. We hypothesized that CXCR2 mediates the proangiogenic effects of ELR(+) CXC chemokines during tumorigenesis. To test this postulate, we used syngeneic murine Lewis lung cancer (LLC; 3LL, H-2(b)) heterotopic and orthotopic tumor model systems in C57BL/6 mice replete (CXCR2(+/+)) and deficient in CXCR2 (CXCR2(-/-)). We first demonstrated a correlation of the expression of endogenous ELR(+) CXC chemokines with tumor growth and metastatic potential of LLC tumors. Next, we found that LLC primary tumors were significantly reduced in growth in CXCR2(-/-) mice. Moreover, we found a marked reduction in the spontaneous metastases of heterotopic tumors to the lungs of CXCR2(-/-) mice. Morphometric analysis of the primary tumors in CXCR2(-/-) mice demonstrated increased necrosis and reduced vascular density. These findings were further confirmed in CXCR2(+/+) mice using specific neutralizing Abs to CXCR2. The results of these studies support the notion that CXCR2 mediates the angiogenic activity of ELR(+) CXC chemokines in a preclinical model of lung cancer.     

Nonredundant function of phosphodiesterases 4D and 4B in neutrophil recruitment to the site of inflammation

Neutrophils have been implicated in the pathogenesis of many inflammatory lung diseases, including chronic obstructive pulmonary disease and asthma. With this study, we investigated how disruption of cAMP signaling impacts the function of neutrophil recruitment to the lung. Four genes code for type 4 phosphodiesterases (PDE4s), enzymes critical for regulation of cAMP levels and cell signaling. Ablation of two of these genes, PDE4B and PDE4D, but not PDE4A, has profound effects on neutrophil function. In a paradigm of mouse lung injury induced by endotoxin inhalation, the number of neutrophils recovered in the bronchoalveolar lavage was markedly decreased in PDE4D(-/-) and PDE4B(-/-) mice 4 and 24 h after exposure to LPS. Acute PDE4 inhibition with rolipram had additional inhibitory effects on neutrophil migration in PDE4B(-/-) and, to a lesser extent, PDE4D(-/-) mice. This decreased neutrophil recruitment occurred without major changes in chemokine accumulation in bronchoalveolar lavage, suggesting a dysfunction intrinsic to neutrophils. This hypothesis was confirmed by investigating the expression of adhesion molecules on the surface of neutrophils and chemotaxis in vitro. CD18 expression was decreased after ablation of both PDE4B and PDE4D, whereas CD11 expression was not significantly affected. Chemotaxis in response to KC and macrophage inflammatory protein-2 was markedly reduced in PDE4B(-/-) and PDE4D(-/-) neutrophils. The effect of PDE4 ablation on chemotaxis was comparable, but not additive, to the effects of acute PDE4 inhibition with rolipram. These data demonstrate that PDE4B and PDE4D play complementary, but not redundant, roles in the control of neutrophil function.     

Protection of Xenopus laevis embryos against alcohol-induced delayed gut maturation and growth retardation by peroxiredoxin 5 and catalase

Accumulated evidence indicates that maternal alcohol consumption causes fetal enteric damage and growth retardation. In this study, we investigated the underlying molecular mechanisms in a Xenopus model of fetal alcohol exposure. We established a condition of transient alcohol exposure that produces tadpoles with delayed gut maturation and decreased body length. We then investigated the roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by microinjecting plasmids expressing catalase and peroxiredoxin 5 (PRDX5) into two-cell stage embryos. Finally, the effects of these enzymes on the expression of key gut developmental genes were determined by animal cap explant assay. We showed that exposure of Xenopus embryos to 0.5% alcohol from stage 13 to stage 22 produced tadpoles with delayed gut maturation, reduced growth, and down-regulation in several gut developmental genes, with VegT, Pax6 and Sox17 most vulnerable. We further demonstrated that microinjection of catalase attenuated alcohol-induced ROS production and restored the expression of VegT and Pax6, but protected the embryos from delayed gut development and retarded growth only partially. By contrast, microinjection of PRDX5 reduced both ROS and RNS production, and prevented the gut and growth defects, and restored VegT, Pax6 and Sox17 gene expression. A positive correlation was found between delayed gut maturation and reduced body length. These results indicate the crucial roles of both the ROS-Pax6 and RNS-Sox17 signaling axes in alcohol-induced fetal gut defects and growth retardation. In addition, they suggest strongly a cause-and-effect relationship between alcohol-induced delayed gut maturation and growth retardation.