Sphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. The lipid second messenger sphingosine 1-phosphate (S1P) 2 has been implicated in the regulation of a variety of important mammalian cell processes, including proliferation, differentiation, and apoptosis (1-3). Interest in S1P has focused recently on two distinct cellular actions of this lipid, namely the function of S1P as an extracellular ligand activating specific G protein-coupled receptors and the role of S1P as an intracellular second messenger (4). Noticeably, some of the diverse signaling roles attributed to elevated cellular S1P levels include prevention of ceramide-induced apoptosis (5, 6) and calcium mobilization (7).Sphingosine kinase (SPHK), the enzyme that catalyzes the phosphorylation of sphingosine, plays a central role in the regulation of intracellular levels of S1P. Two isoforms of mammalian SPHK (SPHK1 and SPHK2) have been cloned and characterized (8, 9). SPHK1 is a cytosolic enzyme with an apparent molecular mass of 49 kDa and contains five conserved domains, the second of which has a conserved ATP binding motif found in diacylglycerol kinases (8). Overexpression of SPHK1 induces cell proliferation by promoting the G 1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (10). SPHK2 contains the same five conserved domains found in SPHK1 while also having divergent sequences at the N-terminal and in the middle regions, resulting in a protein 200 amino acids larger than SPHK1. In addition, heterogeneity in the N terminus was found in SPHK2 (11), whose mechanism of generation remains unknown. The role of SPHK2, however, has not been elucidated until recently. Studies from our laboratory have demonstrated that SPHK2 is a nuclear protein and inhibits DNA synthesis when overexpressed in mammalian cells (12). Similarly, Liu et al. (13) have reported that SPHK2 induces apoptosis through its putative BH3 domain. More recently, SPHK2 has been postulated to function as a potential immunomodulator either through phosphorylation of an immunosuppressant drug, FTY720 (11,14), or association wit...

Background: Mice deficient in the platelet receptor CLEC-2 for podoplanin showed impaired blood/lymphatic vessel separation. Results: Functions of lymphatic endothelial cells are inhibited by platelet releasates and BMP-9, which we identified as a novel releasate. Conclusion: Granule contents including BMP-9 released upon platelet activation by CLEC-2-podoplanin interaction may contribute to the separation in vivo. Significance: We proposed a novel mechanism of platelet-mediated blood/lymphatic vessel separation.

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, downregulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetateinduced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD overexpression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling. Sphingosine kinases (SPHKs)3 catalyze the formation of sphingosine 1-phosphate, a bioactive lipid that regulates a diverse range of cellular processes, including cell growth, survival, differentiation, motility, and cytoskeletal organization (1, 2). Some of these cellular processes are mediated by five sphingosine 1-phosphatespecific G protein-coupled receptors, whereas others appear to be controlled by intracellular sphingosine 1-phosphate through as yet unidentified intracellular targets (2, 3).Two distinct SPHK isoforms, SPHK1 and SPHK2, have been cloned and characterized (4, 5). Diverse external stimuli, particularly growth and survival factors, stimulate SPHK1, and intracellularly generated sphingosine 1-phosphate has been implicated in their mitogenic and anti-apoptotic effects (6 -15). Expression of SPHK1 enhanced proliferation, promoted the G 1 /S transition, protected cells from apoptosis (6,8,16), and induced tumor formation in mice (8, 9).In contrast to SPHK1 much less is known about SPHK2. Although highly similar in amino acid sequence and possessing five evolutionarily conserved domains found in all SPHKs (17), SPHK2 diverges in its N terminus and central regions. These two isoforms have different kinetic properties and differ in developmental and tissue expression (5) implying that they may have distinct physiological functions. In fact, studies from our laboratory have demonstrated that, in contrast to cytosolic distribution of SPHK1, SPHK2 enters nuclei and inhibits DNA synthesis or induces apoptosis under stressful conditions such as serum deprivation (18,19)....

CLEC-2 has been described recently as playing crucial roles inClec-2 ؉/؉ embryos, we were able to demonstrate that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, possibly through homophilic interactions without apparent increase in bleeding tendency. We propose that CLEC-2 could be an ideal novel target protein for an anti-platelet drug, which inhibits pathological thrombus formation but not physiological hemostasis.

Background: Early mesoderm can be classified into Flk-1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk-1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato-endothelial lineages has not been proved in mouse embryos. Results: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin-Kit+Sca-1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk-1+ population that exists until E8.5 may be the source for hemato-endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin-Kit+Sca-1+ (KSL) cells, respectively. Conclusions: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos. Developmental Dynamics 242:254–268, 2013. © 2013 Wiley Periodicals, Inc.Key FindingsPDGF receptor alpha–positive mesoderm contributes to endothelial and hematopoietic cells in physiological mouse embryogenesis.PDGF receptor alpha–positive mesoderm in early embryo is distinct from yolk sac blood island mesoderm, representing a source of hematopoietic cells on the embryo proper side.Genetic manipulation of Etv2 or Runx1 in PDGF receptor alpha–positive mesoderm demonstrates the functional significance of this mesoderm subset in vascular development and hematopoiesis.

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Salinity stress has become an expanding threat to food security worldwide. Revealing the mechanisms of salinity tolerance in plants has immense significance. Foxtail millet (Setaria italica L.) has been regarded as a model crop for exploring mechanisms under stress, considering its extreme adaptation abilities to adverse ecologies. in present study, two foxtail millet cultivars of Yugu2 and An04 with contrasting salt tolerance properties were investigated through integrative analyses of transcriptomics and metabolomics. In the transcriptomics results, 8887 and 12,249 DEGs were identified in Yugu2 and An04 in response to salinity, respectively, and 3149 of which were overlapped between two varieties. these salinity-responsive genes indicated that ion transport, redox homeostasis, phytohormone metabolism, signaling and secondary metabolism were enriched in Yugu2 by GO and KEGG analyses. The integrative omics analysis implied that phenylpropanoid, flavonoid and lignin biosynthesis pathways, and lysophospholipids were vital in determining the foxtail millet salinity tolerance. Importantly, the tolerance of Yugu2 attributed to higher efficiencies of ion channel and antioxidant system. All these provide a comprehensive regulatory network of foxtail millet to cope with salinity, and shed some lights on salt tolerance which is relevant for other cereal crops. Soil salinity is a growing problem for irrigated agriculture, and it severely limits the productivity and geographical distribution of crops. About 800 × 10 6 million ha of land is affected by salinity with an annual increase of ∼1-2% worldwide 1. High salinity first leads to osmotic stress and then ion toxicity to plants, with detrimental effects in inhibiting all the major physiological processes such as germination, growth and morphogenesis, photosynthesis, nutrient absorption, and yield of plants 2. To mount an effective response to cope with salinity, plants have evolved abilities to sense and manage both the osmotic stress and ions toxicity. The sensory modality of plants in response to NaCl was distinct from the response to purely osmotic stress 3. However, it is not completely understood whether plants have receptor or sensor for Na +4. As per reported, Na + enter cytoplasm probably via various cation exchange channels, such as

Hematopoietic cells (HPCs) develop from hemogenic endothelial cells (ECs), a specialized type of ECs undergoing hematopoietic transition. However, the mesoderm origin for hemogenic ECs or HPCs has not been clarified. To examine the origin for hemogenic mesoderm, we inactivated Etv2, a master regulator for EC/HPC commitment, in specific regions. Region-specific Etv2 ablation in early mesoderm caused local EC differentiation block, resulting in the loss of specific vascular beds without compensatory migration of residual ECs into avascular area. This feature of local EC/HPC differentiation block was correlated to the hemogenic potential of each mesoderm subset. We found that caudal-lateral mesoderm of E7.5-8.5 embryos represent the pre-committed population critical for generating hemogenic ECs. Etv2 ablation in caudal-lateral mesoderm by Hoxb6 Cre or Hoxb6CreER transgene affected vitelline plexus formation and intra-aortic hematopoietic clusters. In differentiated embryonic stem cells, this mesoderm subset marked by Hoxb6-lateral mesoderm promoter showed enriched T lymphopoietic potential among Flk-1(+) cells, which could be regarded as a characteristic for definitive HPCs. These findings indicate that critical mesoderm precursors possibly for definitive type hemogenic ECs are regionally specified in primitive mesoderm, suggesting that Hoxb6(+) caudal-lateral mesoderm represents the critical source of HPCs, which are potentially useful to enrich definitive HPCs from embryonic stem cells.