Isolation of human promoter regions by Alu repeat consensus-based polymerase chain reaction.

Knowledge of the promoter structure is critical for an understanding of the regulation of genes. We demonstrate by analysis of 405 human genes that human promoter regions are flanked by upstream Alu repeat elements, typically at a distance of 0.5-5 kb from their protein-coding areas. We identified common Alu repeat consensus sequences (ARC) among the different members of the Alu subfamilies that can be used as universal anchor sites for polymerase chain reaction (PCR) amplification. Utilizing ARC-specific primers and oligonucleotides specific for the 5' end of a selected target gene, we show that sequences spanning unknown human gene promoter regions can be directly amplified by PCR from genomic DNA. This novel technique, termed ARC-PCR, allowed us to characterize the proximal promoters of the human LTA4 hydrolase and SPARC genes, each within 1 day.

IGF-1, PDGF and CD18 are adherence-responsive genes: regulation during monocyte differentiation.

Unstimulated mononuclear cells express IGF-1, PDGF-A and PDGF-B mRNA, but not a number of other genes coding for growth factors or cytokines, as we demonstrated previously. The main focus of the present investigation was to compare gene expression of mononuclear cells unstimulated in suspension with gene expression of monocytes stimulated by adherence. mRNA levels of IGF-1-A and -B, PDGF-A, -B, PD-ECGF, basic FGF, acidic FGF, TGF-alpha, TGF-beta 1, and IGF-2 were sought for and quantified with our sensitive RT-PCR method (3n-PCR). The respective mRNAs of basic FGF, acidic FGF, TGF-alpha and IGF-2 were not detected, independent of the culture conditions. In suspension culture, mRNA levels of IGF-1A and -B, PDGF-A, -B, and CD18 remained unchanged. Monocyte adherence regulated IGF-1A, PDGF-A, and -B mRNA levels. In parallel, mRNA levels of the monocyte adhesion molecule CD18 increased rapidly (4.5-fold). In contrast, independent of the presence of an adherence stimulus, the mRNAs for the cytoskeletal structure protein beta-actin and PD-ECGF remained constant, whereas mRNA for growth factors TGF-beta 1 and IGF-1B, respectively, was increased. Thus, monocyte adherence selectively regulates IGF-1, PDGF-A, PDGF-B and CD18 mRNAs (adherence-responsive genes) in a coordinated manner. This led us to identify two novel consensus elements within their respective functional promoters. Both motifs, an 11 bp purine-rich sequence and a 13 bp pyrimidine-rich segment, respectively, are absent from the genes that were not specifically activated by adherence. The identified elements are potential binding sites for transcription factors that may define a common basis for the regulation of the adherence-responsive genes IGF-1A, PDGF-A, PDGF-B and CD18.

Regulation of angiogenesis by SPARC and angiostatin: implications for tumor cell biology.

The formation of blood vessels through endothelial cell proliferation from extant vasculature (angiogenesis) is a prerequisite for the remodeling, regeneration and repair of tissue. In pathological processes, angiogenesis is a major limiting step in tumor growth and is necessary for tumor formation at metastatic sites. Angiogenesis consists of a sequence of events that include (i) dissolution of the basement membrane, (ii) migration and (iii) proliferation of endothelial cells, (iv) formation of the vascular loop, and (v) formation of a new basement membrane. A variety of factors control and regulate these multiple steps during which the endothelium gives rise to new vessels. Growth factors such as vascular endothelial growth factor or platelet-derived growth factor act directly on endothelial cells and/or activate inflammatory cells (monocytes and T lymphocytes) which in turn synthesize angiogenic factors. SPARC (Secreted Protein Acidic and Rich in Cytsteine), in conjunction with other known angiogenic factors, might act pleiotropically during angiogenesis. Recently, angiostatin, a novel endogenous inhibitor of metastasis, has been reported. This protein, a cleavage product of plasminogen, most likely acts through inhibition of angiogenesis. Current research is focused on the angiogenic and antiangiogenic properties of SPARC and angiostatin and will provide us with a better understanding of how these important factors regulate angiogenesis.

The human leukotriene A4 hydrolase gene is expressed in two alternatively spliced mRNA forms.

We identified a novel short (s-) mRNA of the human leukotriene-A4 (LTA4) hydrolase using sequential reverse transcriptase PCR mapping. The s-mRNA is generated by skipping an 83 bp exon located in the 3' coding region and suggests the expression of an LTA4 hydrolase isoform with a calculated molecular mass of 59 kDa and a distinct C-terminus. Both LTA4 hydrolase mRNAs are constitutively expressed in blood cells, endothelial cells, smooth muscle cells, fibroblasts and tumour cells. The ratios of their mRNA expression levels are cell specific, with relatively high s-form mRNA expression in reticulocytes. Our data strongly suggest that the novel mRNA codes for the structurally related but distinct LTA4 hydrolase isoenzyme that has been postulated.

Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes.

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12-LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

Thrombosis and inflammation as multicellular processes: significance of cell-cell interactions.

Platelet activation as a result of vascular injury provokes endothelial cells to respond in a manner which limits or reverses the occlusive consequences of platelet accumulation. If the agonistic forces are strong, platelet accumulation is irreversible. In vitro data from our laboratory have repeatedly demonstrated that platelets become unresponsive to all agonists when in proximity to endothelial cells. This unresponsiveness is due to at least three separate endothelial "thromboregulatory" systems: eicosanoids, endothelium-derived relaxing factor (EDRF/NO), and most importantly an endothelial cell ecto-nucleotidase which metabolizes released platelet adenosine diphosphate (ADP) with consequent restoration of platelets to the resting state. This nucleotidase is operative in the complete absence of EDRF/NO and eicosanoids, indicating that the latter two are dispensable thromboregulators. We have solubilized the human endothelial cell ectoADPase, as well as that from placental tissue. Candidate proteins from a purified ADPase fraction are now being studied in further detail. An understanding of the molecular biology of the ADPase gene may lead to development of therapeutic agents such as soluble forms of the enzyme as well as approaches toward up-regulation of ectoADPase activity. This could result in "early thromboregulation", i.e. prevention and/or reversal of platelet accumulation at sites of vascular damage via immediate metabolic removal of the prime platelet agonist-ADP.

Growth factor mRNA profiles in unstimulated human mononuclear cells: identification of genes which are constitutively and variably expressed.

Quiescent human mononuclear cells (MNC) as studied ex vivo express highly specific mRNA patterns of growth factors: We recently demonstrated that unstimulated MNC constitutively express the genes for the A and B chains of platelet-derived growth factor (PDGF). This expression was down-regulated by dietary omega-3 fatty acids. We now report that unstimulated human MNC express the genes for platelet-derived endothelial cell growth factor (PD-ECGF), insulin-like growth factor (IGF-1A, -1B) and transforming growth factor-beta 1 (TGF-beta 1). In contrast, acidic and basic fibroblast GF (FGFs), insulin-like GF-2 (IGF-2), transforming GF-alpha (TGF-alpha) and epidermal GF (EGF) were not expressed in MNC, nor were alpha- and beta- receptors for PDGF. Quantitatively, as measured over a period of six weeks, expression of PD-ECGF was constant, whereas TGF-beta 1, IGF-1A, and IGF-1B were expressed at varying levels and all independently of each other. Dietary omega-3 fatty acids had no effect on gene expression. Our results also indicate that down-regulation of PDGF gene expression represents a specific and possibly therapeutic effect of dietary fish oil supplementation.

Dietary omega-3 fatty acids lower levels of platelet-derived growth factor mRNA in human mononuclear cells.

Platelet-derived growth factor (PDGF) is a potent mitogen thought to propagate atherosclerosis and other proliferative or inflammatory diseases. Some of these diseases are ameliorated in humans by ingestion of omega-3 fatty acids. We investigated mRNA expression of both PDGF-A and PDGF-B in quiescent peripheral blood mononuclear cells from healthy male volunteers. For this, a highly sensitive, quantitative polymerase chain reaction strategy (3n-PCR) was developed. In contrast to granulocytes, both PDGF-A and PDGF-B mRNAs are expressed in mononuclear cells. This expression occurs at a remarkably constant rate. Moreover, effects of 7 g/d of a 85% omega-3 fatty acid fish oil concentrate were investigated in a 6-week controlled, randomized, observer-blind study in 14 human volunteers, 7 of whom served as controls. omega-3 Fatty acids increased in mononuclear cell phospholipids. We demonstrate for the first time that diet affects human gene regulation. Dietary omega-3 fatty acids downregulate gene expression of both PDGF-A (-66%), and PDGF-B (-70%). This may represent a novel mechanism for the antifibrotic and antiatherosclerotic action of omega-3 fatty acids.

n-3 fatty acids and cysteinyl-leukotriene formation in humans in vitro, ex vivo, and in vivo.

In humans, dietary n-3 fatty acids ameliorate some chronic inflammatory diseases. In contrast, however, dietary n-3 fatty acids had no effect in patients with bronchial asthma. In bronchial asthma, the cysteinyl-leukotrienes C4, D4, and E4, formed from arachidonic acid, are considered important mediators. They are as vasoconstrictive and bronchoconstrictive as leukotrienes C5, D5, and E5, cysteinyl-leukotrienes derived from eicosapentaenoic acid. Whether and how n-3 fatty acids affect human cysteinyl-leukotriene metabolism is largely unknown. We therefore investigated human cysteinyl-leukotriene metabolism in vitro, ex vivo, and in vivo in the absence and presence of dietary n-3 fatty acids. We demonstrate formation of leukotrienes C5, D5, and E5 from eicosapentaenoic acid in vitro and ex vivo in stimulated human granulocytes. Proof of formation relies on cochromatography with authentic standards on reverse-phase high-performance liquid chromatography, ultraviolet-absorbance spectra, and radioactive tracer studies. In vitro, amounts of leukotrienes C5, D5, and E5 formed depended on the amount of exogenous eicosapentaenoic acid; leukotrienes C4, D4, and E4 formed from endogenous arachidonic acid, however, remained unaltered. A randomized, controlled, observer-blind study in 14 human volunteers, seven of whom supplemented their diet with 7 gm/day of an 85% n-3 fatty acid concentrate for 6 weeks was subsequently performed. Ex vivo, levels of leukotriene E5 almost equaled those of leukotriene E4. Moreover, urinary excretion of leukotriene E4 was assessed to estimate formation of cysteinyl leukotrienes from arachidonic acid in vivo. Urinary excretion of leukotriene E4 was reduced by 35% after dietary supplementation with n-3 fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and sequencing of a murine pgk-1 pseudogene family.

Seven genomic mouse DNA fragments carrying pgk-1-homologous regions have been cloned and sequenced. They have to be classified as processed genes because intervening sequences, present in their productive counterpart, are absent. Four pseudogenes (I-IV) represent nearly the complete sequence of pgk-1 cDNA. Two of these genes (I and II), although rather different from the published mouse pgk-1 cDNA in the 3'-untranslated region, represent the actual mouse pgk-1 cDNA sequence in the coding part except for substitutions in the third position of three codons. These genes can code for a functional PGK protein but, lacking as they do classical promoter structures, are probably not expressed. They show the typical characteristics of retroposons, being flanked by A-rich regions and direct repeats which are localized at the positions where the homology with the mouse pgk-1 cDNA is interrupted. Pseudogenes III and IV have numerous mutations. Gene III is also flanked by direct repeats, whereas gene IV is flanked by inverted repeats. The other three genes are flanked by direct repeats localized further inside the target sites. They are truncated and mutated extensively as usually observed with pseudogenes.