Targeted disruption of the ABP-120 gene leads to cells with altered motility

The actin-binding protein ABP-120 has been proposed to play a role in cross-linking F-actin filaments during pseudopod formation in motile Dictyostelium amebas. We have tested this hypothesis by analyzing the phenotype of mutant cell lines which do not produce ABP-120. Two different transformation vectors capable of targeted disruption of the ABP-120 gene locus have been constructed using a portion of an ABP-120 cDNA clone. Three independent cell lines with different disruption events have been obtained after transformation of amebas with these vectors. The disruption of the ABP-120 gene by vector sequences results in either the production of a small amount of truncated ABP-120 or no detectable protein at all. The phenotypes of two different clones lacking ABP-120, generated in strains AX3 and AX4, have been characterized and show identical results. ABP-120- cells tend to remain rounder before and after cAMP stimulation, and do not reextend pseudopods normally after rapid addition of cAMP. In addition, ABP-120- cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. The amount of F-actin cross-linked into the cytoskeleton after cAMP stimulation of ABP-120- cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton, and this correlates temporally with the absence of reextension of pseudopods after cAMP stimulation. The instantaneous velocity is significantly reduced both before and after cAMP stimulation in the ABP-120- cells, and the cells show decreased chemotactic efficiency compared to ABP-120+ controls. This phenotype is consistent with a role for ABP-120 in pseudopod extension by cross-linking actin filaments as proposed by the "cortical expansion model" (Condeelis, J., A. Bresnick, M. Demma, C. Dharmawardhane, R. Eddy, A. L. Hall, R. Sauterer, and V. Warren. 1990. Dev. Genet. 11:333-340).     

SNARE complex-mediated degranulation in mast cells

Mast cell function and dysregulation is important in the development and progression of allergic and autoimmune disease. Identifying novel proteins involved in mast cell function and disease progression is the first step in the design of new therapeutic strategies. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins demonstrated to mediate the transport and fusion of secretory vesicles to the membrane in mast cells, leading to the subsequent release of the vesicle cargo through an exocytotic mechanism. The functional role[s] of specific SNARE family member complexes in mast cell degranulation has not been fully elucidated. Here, we review recent and historical data on the expression, formation and localization of various SNARE proteins and their complexes in murine and human mast cells. We summarize the functional data identifying the key SNARE family members that appear to participate in mast cell degranulation. Furthermore, we discuss the utilization of RNA interference (RNAi) methods to validate SNARE function and the use of siRNA as a therapeutic approach to the treatment of inflammatory disease. These studies provide an overview of the specific SNARE proteins and complexes that serve as novel targets for the development of new therapies to treat allergic and autoimmune disease.     

Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption.

Semaphorin 3C is a secreted member of the semaphorin gene family. To investigate its function in vivo, we have disrupted the semaphorin 3C locus in mice by targeted mutagenesis. semaphorin 3C mutant mice die within hours after birth from congenital cardiovascular defects consisting of interruption of the aortic arch and improper septation of the cardiac outflow tract. This phenotype is similar to that reported following ablation of the cardiac neural crest in chick embryos and resembles congenital heart defects seen in humans. Semaphorin 3C is expressed in the cardiac outflow tract as neural crest cells migrate into it. Their entry is disrupted in semaphorin 3C mutant mice. These data suggest that semaphorin 3C promotes crest cell migration into the proximal cardiac outflow tract.     

Thrombin-induced degranulation of cultured bone marrow-derived mast cells

This study was undertaken to determine if a plasma protease such as thrombin, a highly specific procoagulant enzyme that has numerous effects on platelets, endothelial cells, and smooth muscle cells, could mediate mast cell activation processes. Our results indicate that at near physiologic levels, thrombin can rapidly trigger mast cell degranulation without activating the 5-lipoxygenase system.     

Mycophenolic acid inhibits the degranulation of rat peritoneal mast cells.

The effect of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase, on the degranulation of rat peritoneal mast cells (RMC) was studied. RMC were pretreated for 48 hr with 0.1-10 microM MPA before the cells were sensitized with IgE and triggered with specific Ag. The net amount of [3H]5-HT released from granules was decreased by 44 and 32% with 1 and 10 microM MPA treatment, respectively. MPA inhibition of degranulation was completely reversed by the addition of 30 microM guanosine to the incubation medium. There was no difference in the apparent number or affinity of IgE binding sites between control and MPA-treated RMC. MPA pretreatment also had no effect on the IgE receptor-mediated production of PGD2 in RMC. These results suggest that depletion of intracellular GTP pools by MPA can disrupt the signaling between the IgE receptor and the secretory granules and that, under these same conditions, the release and metabolism of arachidonic acid are unaffected.     

Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal transduction pathways.

The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.     

Targeted disruption of the tyrosine phosphatase PTPalpha leads to constitutive downregulation of the kinases Src and Fyn.

A role for the receptor-like protein tyrosine phosphatase alpha (PTPalpha) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPalpha enhances the dephosphorylation and activation of Src and Fyn [1] [2] [3]. We have generated mice lacking catalytically active PTPalpha to address the question of whether PTPalpha is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPalpha allele (PTPalpha-/-) and lacking detectable PTPalpha protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPalpha-/- mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPalpha-/- mice. Thus, PTPalpha is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPalpha-/- mouse brain lysates. These may be PTPalpha substrates or downstream signaling proteins. Taken together, the results indicate that PTPalpha has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.     

Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.     

Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice

DNA ligase IV is the most recently identified member of a family of enzymes joining DNA strand breaks in mammalian cell nuclei [1] and [2]. The enzyme occurs in a complex with the XRCC4 gene product [3], an interaction mediated via its unique carboxyl terminus [4] and [5]. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination [3] and [6], implicating DNA ligase IV in the pathway of nonhomologous end-joining (NHEJ) of DNA double-strand breaks mediated by XRCC4, the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in mammalian cells (reviewed in [7]). The phenotype of a null mutant of the Saccharomyces cerevisiae DNA ligase IV homologue indicates that the enzyme is non-essential and functions in yeast NHEJ [8], [9] and [10]. Unlike other mammalian DNA ligases for which cDNAs have been characterised, DNA ligase IV is encoded by an intronless gene (LIG4). Here, we show that targeted disruption of LIG4 in the mouse leads to lethality associated with extensive apoptotic cell death in the embryonic central nervous system. Thus, unlike Ku70/80 and DNA-PKcs [11], [12], [13] and [14], DNA ligase IV has an essential function in early mammalian development.     

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.     

Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium

Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca²⁺ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10⁻⁴–10⁻⁵ M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.     

An unexpected role for autophagy in degranulation of mast cells

Mast cells play a crucial role in allergic inflammatory reactions through releasing cytosolic granules upon antigen stimulation. However, the mechanisms underlying maturation and release of secretory granules are not fully understood. We found that autophagy is constitutively induced in mast cells under full nutrition conditions, and type II LC3 (LC3-II), a marker for autophagosomes, localizes on secretory granules. While deletion of Atg7 does not impair the development of bone marrow-derived mast cells (BMMCs), Atg7-deficient BMMCs show severe impairment of degranulation, but not cytokine production, upon antigen stimulation. Moreover we found that LC3-II, but not LC3-I, colocalizes with CD63, a marker for secretory lysosomes and is released extracellularly along with degranulation in wild-type BMMCs, but not Atg7-deficient BMMCs. Finally, passive cutaneous anaphylaxis reactions are almost completely abolished in mast celldeficient mice reconstituted with Atg7-deficient BMMCs. Collectively, these results suggest that autophagy is not essential for the development, but plays a crucial role in degranulation, of mast cells.     

Substance P induces granulocyte infiltration through degranulation of mast cells

Substance P, a potent vasodilatory neuropeptide, is released from peripheral nerve endings of sensory neurons by various stimuli. Although in vitro incubation of rat and human mast cells with substance P causes their degranulation, it is not known whether inflammatory changes induced by substance P are mediated by degranulation of mast cells. We investigated this point by using genetically mast cell-deficient WBB6F1-W/Wv and WCB6F1-Sl/Sld mice. The s.c. injection of substance P induced degranulation of mast cells in the skin of WBB6F1-+/+ mice, and then a marked eosinophil infiltration around the degranulated mast cells. However, WBB6F1-W/Wv and WCB6F1-Sl/Sld mice showed little or no eosinophil infiltration in the skin after the injection of substance P. When the mast cell deficiency of WBB6F1-W/Wv mice was rescued either systemically by bone marrow transplantation or locally by injection of cultured mast cells, injection of substance P induced the infiltration of eosinophils, suggesting that substance P-induced eosinophil infiltration was mediated through degranulation of mast cells.     

An unexpected role for autophagy in degranulation of mast cells

Mast cells play a crucial role in allergic inflammatory reactions through releasing cytosolic granules upon antigen stimulation. However, the mechanisms underlying maturation and release of secretory granules are not fully understood. We found that autophagy is constitutively induced in mast cells under full nutrition conditions, and type II LC3 (LC3-II), a marker for autophagosomes, localizes on secretory granules. While deletion of Atg7 does not impair the development of bone marrow-derived mast cells (BMMCs), Atg7-deficient BMMCs show severe impairment of degranulation, but not cytokine production, upon antigen stimulation. Moreover we found that LC3-II, but not LC3-I, colocalizes with CD63, a marker for secretory lysosomes and is released extracellularly along with degranulation in wild-type BMMCs, but not Atg7-deficient BMMCs. Finally, passive cutaneous anaphylaxis reactions are almost completely abolished in mast celldeficient mice reconstituted with Atg7-deficient BMMCs. Collectively, these results suggest that autophagy is not essential for the development, but plays a crucial role in degranulation, of mast cells.     

Targeted disruption of MAIL, a nuclear IkappaB protein, leads to severe atopic dermatitis-like disease

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IkappaB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor kappaB (IkappaB) zeta. In this study, we generated Mail-/- mice to investigate the roles of MAIL in whole organisms. Mail-/- mice grew normally until 4-8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail-/- mice than in normal. Histopathological analysis indicated that the Mail-/- skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail-/- skin lesions, similar to that observed in the skin of patients with AD. In Mail-/- mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail-/- mouse is a valuable new animal model for research on AD.     

The effect of cyclic AMP on the maturation and degranulation of mast cells.

In short-term tissue cultures dibutyryl cAMP inhibits the cortisone-induced degranulation of mast cells. The effect of methylxanthine was found to be similar but developed at a slower rate. The increase of cAMP level also affected the maturation of mast cells, as the ratio of cells of mixed granulation increased, compared to the alcian blue- and safranin-present model inhibited degranulation.     

Targeted disruption of CRE-binding factor TREB5 gene leads to cellular necrosis in cardiac myocytes at the embryonic stage.

TREB5 (hXBP-1) is a basic region leucine zipper protein which binds to a CRE-like element in both human T-cell leukemia virus type 1 and MHC class II genes. To study the function(s) of TREB5 in normal development, we have generated TREB5 deficient mice by gene targeting. Heterozygous mutant mice have not exhibited any obvious abnormalities; however, homozygous mutant embryos die between embryonic days 10.5 and 14.5. The major defect responsible for lethality is cellular necrosis of cardiac myocytes located at the atrium and the truncus arteriosus with its following ventricle. Necrotic alteration was not observed in either the endocardial cushion or the conotruncal ridge. These results indicate that TREB5 plays an essential role in maintenance and/or growth of cardiac myocytes during cardiogenesis.     

Ceramide kinase is a mediator of calcium-dependent degranulation in mast cells

Ceramide kinase (CERK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P) and is known to be activated by calcium. Although several groups have examined the functions of CERK and its product C1P, the functions of C1P and CERK are not understood. We studied the RBL-2H3 cell line, a widely used model for mast cells, and found that CERK and C1P are required for activation of the degranulation process in mast cells. We found that C1P formation was enhanced during activation induced by IgE/antigen or by Ca(2+) ionophore A23187. The formation of C1P required the intracellular elevation of Ca(2+). We generated RBL-2H3 cells that stably express CERK, and when these cells were treated with A23187, a concomitant C1P formation was observed and degranulation increased 4-fold, compared with mock transfectants. The cell-permeable N-acetylsphingosine (C(2)-ceramide), a poor substrate of CERK, inhibited both the formation of C1P and degranulation, indicating that C1P formation was necessary for degranulation. Exogenous introduction of CERK into permeabilized RBL-2H3 cells caused degranulation. We identified a cytosolic localization of CERK that provides exposure to cytosolic Ca(2+). Taken together, these results indicate that C1P formation is a necessary step in the degranulation pathway in RBL-2H3 cells.