Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.     

Interaction with eIF5B is essential for Vasa function during development

The DEAD-box RNA helicase Vasa (Vas) is required for germ cell development and function, as well as for embryonic somatic posterior patterning. Vas interacts with the general translation initiation factor eIF5B (cIF2, also known as dIF2), and thus may regulate translation of specific mRNAs. In order to investigate which functions of Vas are related to translational control, we have analyzed the effects of site-directed vas mutations that reduce or eliminate interaction with eIF5B. Reduction in Vas-eIF5B interaction during oogenesis leads to female sterility, with phenotypes similar to a vas null mutation. Accumulation of Gurken (Grk) protein is greatly reduced when Vas-eIF5B interaction is reduced, suggesting that this interaction is crucial for translational regulation of grk. In addition, we show that reduction in Vas-eIF5B interaction virtually abolishes germ cell formation in embryos, while producing a less severe effect on somatic posterior patterning. We conclude that interaction with the general translation factor eIF5B is essential for Vas function during development.     

Tamoxifen blocks estrogen-induced B cell maturation but not survival

Estrogen treatment has been shown not only to exacerbate disease activity and accelerate death in spontaneous murine models of lupus but also to induce a lupus-like phenotype in non-spontaneously autoimmune mice. In mice transgenic for the H chain of an anti-DNA Ab, estrogen rescues naive autoreactive B cells that normally are deleted and causes them to mature to a marginal zone phenotype. Estrogen further leads to the activation of this population causing an elevation of serum anti-DNA Ab titers and renal disease. This study was designed to evaluate the therapeutic potential of tamoxifen, a selective estrogen receptor modulator, on estrogen-induced lupus. Mice treated with both estradiol and tamoxifen showed no elevation in anti-DNA Ab titers and consequently no glomerular IgG. The DNA-reactive B cell population that is rescued by estrogen was present in an anergic state in mice treated with both estradiol and tamoxifen. Estradiol enhances transitional B cell resistance to apoptosis and expands the population of marginal zone B cells; tamoxifen did not impede the enhanced resistance to apoptosis, but prevented the development of autoreactive cells as marginal zone B cells. Thus, estrogen-induced autoimmunity proceeds through two distinct molecular pathways, one affecting survival and the other maturation. Activation, but not survival, of autoreactive B cells can be abrogated by tamoxifen. Drugs that modulate even some of the effects of estrogen may be beneficial in patients with lupus. Eventually, understanding the pathways involved in survival and activation of autoreactive B cells will permit the development of therapeutics that target all relevant pathways.     

Annexin A5 is not essential for skeletal development

Annexins are highly conserved proteins that are characterized by their ability to interact with phospholipids in a calcium-dependent manner. Although diverse functions have been ascribed to annexins based on in vitro analyses, their in vivo functions still remain unclear. The intensively studied annexin A5 has been identified by its effects on blood coagulation, and subsequently, its function as a calcium-specific ion channel was described. In vitro experiments and expression studies suggested a potential role of annexin A5 during calcification processes in vivo, especially in endochondral ossification. To gain insights into the relevance of annexin A5 in this process, we generated an annexin A5-deficient mouse mutant. Mice lacking annexin A5 are viable, are fertile, and reveal no significant alterations in the biochemical parameters characteristic for metabolic or functional defects. Neither the development of skeletal elements nor the in vitro calcification properties of isolated chondrocytes is significantly impaired by the absence of annexin A5. Therefore, annexin A5 is dispensable for the formation and maintenance of skeletal elements in the mouse and may possibly be pointing to a compensatory effect of other members from the annexin family due to their high functional and structural similarity.     

Mai1p is essential for maturation of proaminopeptidase I but not for autophagy

We here identify Mai1p, a homologue of the autophagy protein Aut10p, as a novel component essential for proaminopeptidase I (proAPI) maturation under non-starvation conditions. In mai1Delta cells mature vacuolar proteinases are detectable and vacuolar acidification is normal. In mai1Delta cells autophagy occurs, though at a somewhat reduced level. This is indicated by proAPI maturation during starvation and accumulation of autophagic bodies during starvation with phenylmethylsulfonyl fluoride. Homozygous diploid mai1Delta cells sporulate, but with a slightly reduced frequency. Biologically active Ha-tagged Mai1p, chromosomally expressed under its native promoter, is at least in part peripherally membrane-associated. In indirect immunofluorescence it localizes to the vacuolar membrane or structures nearby. In some cells Ha-tagged Mai1p appears concentrated at regions adjacent to the nucleus.     

Stat6-protease but not Stat5-protease is inhibited by an elastase inhibitor ONO-5046

A short isoform of Stat6 (65-kDa Stat6), a product of proteolytic processing by an undefined protease (Stat6-protease) in the nucleus, downregulates Stat6-mediated signaling in mast cells. Similarly, Stat5-mediated signaling is downregulated by Stat5-protease in myeloid progenitors. These proteases share a number of characteristics, including their nuclear localization and susceptibility to protease inhibitors. Here, we further investigated these Stat proteases. Interestingly, the activity of Stat6-protease but not of Stat5-protease was inhibited by ONO-5046, an elastase inhibitor that inhibits the activity of neutrophil elastase (NE) and NE-related protease proteinase 3 (PR3). Although both NE and PR3 were able to cleave Stat6 in vitro, the cleavage sites of Stat6 by NE or PR3 differed from that by Stat6-protease in mast cells. In addition, both NE and PR3 could also cleave Stat5, but they differed from Stat5-protease in myeloid progenitors. These results suggest that Stat6-protease may belong to the elastase family but differs from NE or PR3.     

Venom is beneficial but not essential for development and survival of Nasonia

1. Parasitoid wasps sting and inject venom into arthropod hosts, which alters host metabolism and development while keeping the host alive for several days, presumably to induce benefits for the parasitoid young. 2. This study investigates the consequences of host envenomation on development and fitness of wasp larvae in the ectoparasitoid Nasonia vitripennis, by comparing wasps reared on live unstung, previously stung, and cold‐killed hosts. Developmental arrest and suppression of host response to larvae are major venom effects that occur in both stung and cold‐killed hosts, but not in unstung hosts, whereas cold‐killed hosts lack venom effects that require a living host. Thus, cold‐killed hosts mimic some of the effects of venom, but not others. 3. Eggs placed on live unstung hosts have significantly higher mortality during development; however, successfully developing wasps from these hosts have similar lifetime fecundity to that of wasps from cold‐killed or stung hosts. Therefore, although venom is beneficial, it is not required for wasp survival. 4. While wasps developing on both cold‐killed and stung hosts have similar fitness levels, rearing multiple generations on cold‐killed hosts results in significant fitness reductions of wasps. 5. It is concluded that the largest benefits of venom are induction of host developmental arrest and suppression of host response to larva (e.g. immune responses), although more subtle benefits may accrue across generations or under stressful conditions.     

DDR1 signaling is essential to sustain Stat5 function during lactogenesis

Postnatal development of the mammary gland is achieved by an interplay of endocrine and extracellular matrix-derived signals. Despite intense research, a comprehensive understanding of the temporal and spatial coordination of these hormonal and basement membrane stimuli is still lacking. Here, we address the role of the collagen-receptor DDR1 in integrating extracellular matrix-derived signaling with the lactogenic pathway initiated by the prolactin receptor. We found that stimulation of DDR1-overexpressing mammary epithelial HC11 cells with collagen and prolactin resulted in stronger and more sustained induction of Stat5 phosphorylation as compared to control cells. Enhanced Stat5 activity in HC11-DDR1 cells correlated with increased beta-casein gene expression. In contrast, cells derived from DDR1-null mice showed reduced Stat5 activation upon lactogenic stimulation and completely failed to induce beta-casein expression. The cell-autonomous role of DDR1 in controlling ductal branching and alveologenesis prior to the onset of lactogenesis was corroborated by mammary tissue transplantation experiments. Our results show that aside from hormone- and cytokine receptors, DDR1 signaling establishes a third matrix-derived pathway vital to maintain mammary gland function.     

Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1-/- embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of -/- embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1-/- embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.     

MEKK3 is required for endothelium function but is not essential for tumor growth and angiogenesis

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) plays an essential role in embryonic angiogenesis, but its role in tumor growth and angiogenesis is unknown. In this study, we further investigated the role of MEKK3 in embryonic angiogenesis, tumor angiogenesis, and angiogenic factor production. We found that endothelial cells from Mekk3-deficient embryos showed defects in cell proliferation, apoptosis, and interactions with myocardium in the heart. We also found that MEKK3 is required for angiopoietin-1 (Ang1)-induced p38 and ERK5 activation. To study the role of MEKK3 in tumor growth and angiogenesis, we established both wild-type and Mekk3-deficient tumor-like embryonic stem cell lines and transplanted them subcutaneously into nude mice to assess their ability to grow and induce tumor angiogenesis. Mekk3-deficient tumors developed and grew similarly as control Mekk3 wild-type tumors and were also capable of inducing tumor angiogenesis. In addition, we found no differences in the production of VEGF in Mekk3-deficient tumors or embryos. Taken together, our results suggest that MEKK3 plays a critical role in Ang1/Tie2 signaling to control endothelial cell proliferation and survival and is required for endothelial cells to interact with the myocardium during early embryonic development. However, MEKK3 is not essential for tumor growth and angiogenesis. cardiovascular; mitogen-activated protein kinase; embryonic development     

Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development

During vertebrate retinal development, the seven retinal cell types differentiate sequentially from a single population of retinal progenitor cells (RPCs) and organize themselves into a distinct laminar structure. The purpose of this study was to determine whether beta-catenin, which functions both as a nuclear effector for the canonical Wnt signaling pathway and as a regulator of cell adhesion, is required for retinal neurogenesis or lamination. We used the Cre-loxP system to either eliminate beta-catenin or to express a constitutively active form during retinal neurogenesis. Eliminating beta-catenin did not affect cell differentiation, but did result in the loss of the radial arrangement of RPCs and caused abnormal migration of differentiated neurons. As a result, the laminar structure was massively disrupted in beta-catenin-null retinas, although all retinal cell types still formed. In contrast to other neural tissues, eliminating beta-catenin did not significantly reduce the proliferation rate of RPCs; likewise, activating beta-catenin ectopically in RPCs did not result in overproliferation, but loss of neural retinal identity. These results indicate that beta-catenin is essential during retinal neurogenesis as a regulator of cell adhesion but not as a nuclear effector of the canonical Wnt signaling pathway. The results further imply that retinal lamination and retinal cell differentiation are genetically separable processes.     

Vitamin E is essential for mouse placentation but not for embryonic development itself

Vitamin E (alpha-tocopherol) was discovered 80 years ago to be an indispensable nutrient for reproduction in the female. However, it has not been clarified when or where vitamin E is required during pregnancy. We examined the role of alpha-tocopherol in pregnancy using alpha-tocopherol transfer protein (Ttpa)-deficient mice fed specific alpha-tocopherol diets that led to daily, measurable change in plasma alpha-tocopherol levels from nearly normal to almost undetectable levels. A dietary supplement of alpha-tocopherol to pregnant Ttpa-/- (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial development and maturation of the placenta. In addition, exposure to a low alpha-tocopherol environment after initiation of placental formation might result in necrosis of placental syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa(-/-)-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa-/- fetuses were below the detection limit but the fetuses grew normally. These results indicate that alpha-tocopherol is indispensable for the proliferation and/or function of the placenta but not necessary for development of the embryo itself.     

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function.

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.     

Clathrin is spindle-associated but not essential for mitosis

Background

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

Methodology/Principal Findings

Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

Conclusions/Significance

We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.