Expression profiling reveals functionally important genes and coordinately regulated signaling pathway genes during in vitro angiogenesis.

Angiogenesis is a complex multicellular process requiring the orchestration of many events including migration, alignment, proliferation, lumen formation, remodeling, and maturation. Such complexity indicates that not only individual genes but also entire signaling pathways will be crucial in angiogenesis. To define an angiogenic blueprint of regulated genes, we utilized our well-characterized three-dimensional collagen gel model of in vitro angiogenesis, in which the majority of cells synchronously progress through defined morphological stages culminating in the formation of capillary tubes. We developed a comprehensive three-tiered approach using microarray analysis, which allowed us to identify genes known to be involved in angiogenesis and genes hitherto unlinked to angiogenesis as well as novel genes and has proven especially useful for genes where the magnitude of change is small. Of interest is the ability to recognize complete signaling pathways that are regulated and genes clustering into ontological groups implicating the functional importance of particular processes. We have shown that consecutive members of the mitogen-activated protein kinase and leukemia inhibitory factor signaling pathways are altered at the mRNA level during in vitro angiogenesis. Thus, at least for the mitogen-activated protein kinase pathway, mRNA changes as well as the phosphorylation changes of these gene products may be important in the control of blood vessel morphogenesis. Furthermore, in this study, we demonstrated the power of virtual Northern blot analysis, as an alternative to quantitative RT-PCR, for measuring the magnitudes of differential gene expression.

A point mutant of human sphingosine kinase 1 with increased catalytic activity.

Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly(113), a highly conserved residue of human sphingosine kinase 1 (hSK), by site-directed mutagenesis. Surprisingly, a Gly(113)-->Ala substitution generated a mutant that had 1.7-fold greater catalytic activity than wild-type hSK (hSK(WT)). Our data suggests that the Gly(113)-->Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSK(G113A) activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSK(WT).

Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation.

A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.

Time sequence of the inhibition of endothelial adhesion molecule expression by reconstituted high density lipoproteins.

We have used discoidal reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I and dimyristoyl phosphatidylcholine (DMPC) as a tool to investigate the time sequence of the HDL-mediated inhibition of vascular cell adhesion molecule (VCAM)-1 and E-selectin expression in cytokine-activated human umbilical vein endothelial cells (HUVECs). Specifically, we have asked a few questions - (i) how long do the cells need to be exposed to the rHDL before adhesion molecule expression is inhibited and (ii) how long does the inhibition persist after removing the rHDL from the cells. When the cells were not pre-incubated with the rHDL, there was no inhibition. The magnitude of the inhibition increased progressively with increasing duration of pre-incubation up to 16 h. Inhibition did not require the rHDL to be physically present during the activation of adhesion molecule expression by tumour necrosis factor(TNF)-alpha, excluding the possibility that the rHDL was merely interfering with the interaction between TNF-alpha and the cells. When HUVECs were pre-incubated for 16 h with rHDL, the inhibition remained substantial even if the rHDL were removed from the medium up to 8 h prior to addition of TNF-alpha. The HDL-mediated inhibition of VCAM-1 in HUVECs was unaffected by the presence of puromycin, an inhibitor of protein synthesis, excluding the possibility that HDL may have acted by stimulating the synthesis of a cell protein that itself inhibits adhesion molecule expression. These results have important implications in terms of understanding the mechanism(s) of the HDL-mediated inhibition of endothelial adhesion molecule expression.

Nucleotide sequence of the mouse VEGF 3'UTR and quantitative analysis of sites of polyadenylation.

Sequencing of rat and human vascular endothelial growth factor (VEGF) cDNA clones has previously identified a 3' untranslated region of approximately 1.9 kb, although the apparent site of polyadenylation differed in the two species, despite a high degree of sequence conservation in the region. Neither site is preceded by a canonical AAUAAA polyadenylation signal, a situation frequently found in genes that are subject to alternative polyadenylation. We have sequenced 2.25 kb of the 3' region of the mouse VEGF gene and mapped the usage of potential polyadenylation sites in fibroblasts cultured under both normoxic and hypoxic conditions. We find that two sites for polyadenylation are present in the mouse VEGF gene but the majority of transcripts contain the longer form of the 3'UTR and that their usage is not effected by environmental oxygen tension.

Cold shock domain factors activate the granulocyte-macrophage colony-stimulating factor promoter in stimulated Jurkat T cells.

Cold shock domain (CSD) family members have been shown to play roles in either transcriptional activation or repression of many genes in various cell types. We have previously shown that CSD proteins dbpAv and dbpB (also known as YB-1) act to repress granulocyte-macrophage colony-stimulating factor transcription in human embryonic lung (HEL) fibroblasts via binding to single-stranded DNA regions across the promoter. Here we show that the same CSD factors are involved in granulocyte-macrophage colony-stimulating factor transcriptional activation in Jurkat T cells. Unlike the mechanisms of CSD repression in HEL fibroblasts, CSD-mediated activation in Jurkat T cells is not mediated through DNA binding but presumably through protein-protein interactions via the C terminus of the CSD protein with transcription factors such as RelA/NF-kappaB p65. We demonstrate that Jurkat T cells lack truncated CSD factor subtypes present in HEL fibroblasts, which raises the possibility that the cellular content of CSD proteins may determine their final role as activators or repressors of transcription.

An oncogenic role of sphingosine kinase.

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

Reconstitution of T cell-specific transcription directed by composite NFAT/Oct elements.

The complex nature of most promoters and enhancers makes it difficult to identify key determinants of tissue-specific gene expression. Furthermore, most tissue-specific genes are regulated by transcription factors that have expression profiles more widespread than the genes they control. NFAT is an example of a widely expressed transcription factor that contributes to several distinct patterns of cytokine gene expression within the immune system and where its role in directing specificity remains undefined. To investigate distinct combinatorial mechanisms employed by NFAT to regulate tissue-specific transcription, we examined a composite NFAT/AP-1 element from the widely active GM-CSF enhancer and a composite NFAT/Oct element from the T cell-specific IL-3 enhancer. The NFAT/AP-1 element was active in the numerous cell types that express NFAT, but NFAT/Oct enhancer activity was T cell specific even though Oct-1 is ubiquitous. Conversion of the single Oct site in the IL-3 enhancer to an AP-1 enabled activation outside of the T cell lineage. By reconstituting the activities of both the IL-3 enhancer and its NFAT/Oct element in a variety of cell types, we demonstrated that their T cell-specific activation required the lymphoid cofactors NIP45 and OCA-B in addition to NFAT and Oct family proteins. Furthermore, the Oct family protein Brn-2, which cannot recruit OCA-B, repressed NFAT/Oct enhancer activity. Significantly, the two patterns of combinatorial regulation identified in this study mirror the cell-type specificities of the cytokine genes that they govern. We have thus established that simple composite transcription factor binding sites can indeed establish highly specific patterns of gene expression.

Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions.

Inflammation is a basic pathological mechanism that underlies many diseases. An important component of the inflammatory response is the passage of plasma components and leukocytes from the blood vessel into the tissues. The endothelial monolayer lining blood vessels reacts to stimuli such as thrombin or vascular endothelial growth factor by changes in cell-cell junctions, an increase in permeability, and the leakage of plasma components into tissues. Other stimuli, such as tumor necrosis factor-alpha (TNF-alpha), are responsible for stimulating the transmigration of leukocytes. Here we show that angiopoietin-1, a cytokine essential in fetal angiogenesis, not only supports the localization of proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1) into junctions between endothelial cells and decreases the phosphorylation of PECAM-1 and vascular endothelial cadherin, but it also strengthens these junctions, as evidenced by a decrease in basal permeability and inhibition of permeability responses to thrombin and vascular endothelial growth factor. Furthermore, angiopoietin-1 inhibits TNF-alpha-stimulated leukocyte transmigration. Angiopoietin-1 may thus have a major role in maintaining the integrity of endothelial monolayers.

Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 10(6)-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.