The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature.

Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here, we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anticoagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed-specific anticoagulant pathway in microvasculature exposed to low SS.

The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability.

Notch and Angiopoietin-1 (Ang1)/Tie2 pathways are crucial for vascular maturation and stability. Here we identify the transcription factor ERG as a key regulator of endothelial Notch signalling. We show that ERG controls the balance between Notch ligands by driving Delta-like ligand 4 (Dll4) while repressing Jagged1 (Jag1) expression. In vivo, this regulation occurs selectively in the maturing plexus of the mouse developing retina, where Ang1/Tie2 signalling is active. We find that ERG mediates Ang1-dependent regulation of Notch ligands and is required for the stabilizing effects of Ang1 in vivo. We show that Ang1 induces ERG phosphorylation in a phosphoinositide 3-kinase (PI3K)/Akt-dependent manner, resulting in ERG enrichment at Dll4 promoter and multiple enhancers. Finally, we demonstrate that ERG directly interacts with Notch intracellular domain (NICD) and ß-catenin and is required for Ang1-dependent ß-catenin recruitment at the Dll4 locus. We propose that ERG coordinates Ang1, ß-catenin and Notch signalling to promote vascular stability.

Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation.

AIMS: Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide which activates AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated protective genes. METHODS/RESULTS: In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKα(Thr172), and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivation-induced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)(Ser133) phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKα(Thr172) phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression. CONCLUSIONS: These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.

The peroxisomal targeting sequence type 1 receptor, Pex5p, and the peroxisomal import efficiency of alanine:glyoxylate aminotransferase.

Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol. Such apparent import inefficiency is very marked in guinea pig (Cavia porcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homo sapiens) hepatocytes, for example. In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p. Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected Deltapex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e. Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not. In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomyces cerevisiae, whereas AGT-Lys-Lys-Leu was not. These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s. They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu.

Molecular adaptation of alanine:glyoxylate aminotransferase targeting in primates.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is targeted to different organelles (mitochondria and/or peroxisomes) in different species. Possibly under the influence of dietary selection pressure, the subcellular distribution of AGT has changed on at least eight occasions during the evolution of mammals. AGT targeting is dependent on the variable use of two alternative transcription and translation initiation sites which determine whether or not the region encoding the N-terminal mitochondrial targeting sequence is contained within the open reading frame. In the present study, we sequenced the 5' region of the AGT gene, including both ancestral translation start sites, for 11 anthropoid primates and compared the results with data already available for two others. We show that while the more 3' of the two translation start sites is maintained in all species, the more 5' site has been lost in six species (five of seven catarrhines and one of six platyrrhines). In addition, the remaining two catarrhines, which have maintained the 5' translation start site, are predicted to have lost mitochondrial targeting by a different mechanism, possibly loss of the more 5' transcription start site. Analysis of the relative frequencies of nonsynonymous and synonymous mutations in the region encoding the extant or ancestral mitochondrial targeting sequences led us to suggest that there has been recent strong positive selection pressure to lose, or decrease the efficiency of, mitochondrial AGT targeting in several anthropoid lineages, and that the loss of mitochondrial targeting in this group of mammals is likely to have occurred on at least four, and possibly five, separate occasions.

Intracellular localization of dimethylarginine dimethylaminohydrolase overexpressed in an endothelial cell line.

Methylarginines are endogenous inhibitors of nitric oxide synthase (NOS) and have been implicated in the regulation of the nitric oxide pathway in health and disease. Cellular concentrations of free methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH). There are two isoforms of DDAH which have distinct tissue distributions with some relationship to NOS isoforms. We have determined the intracellular localization of both DDAH isoforms by overexpression of epitope-tagged DDAH in an immortalized endothelial cell line. Immunofluorescence confocal microscopy and immunoblotting indicate that both isoforms are predominantly cytosolic with no specific association with organelles or the plasma membrane. These data suggest that the key role for DDAH may be to ensure that under normal conditions the levels of methylarginines are kept low throughout the whole cell.

Evolution of alanine:glyoxylate aminotransferase intracellular targeting: structural and functional analysis of the guinea pig gene.

The distribution of alanine:glyoxylate aminotransferase 1 (AGT) within liver cells has changed many times during mammalian evolution. Depending on the particular species, AGT can be found in mitochondria or peroxisomes, or mitochondria and peroxisomes. In some cases significant cytosolic AGT is also present. In the livers of most rodents, AGT has what is thought to be the more 'ancestral' distribution (i.e. mitochondrial and peroxisomal). However, AGT is distributed very differently in the guinea pig, being peroxisomal and cytosolic. In this study, we have attempted to determine the molecular basis for the loss of mitochondrial AGT targeting and the apparent inefficiency of peroxisomal targeting of AGT in the guinea pig. Our results show that the former is owing to the evolutionary loss of the more 5' of two potential transcription and translation initiation sites, resulting in the loss of the ancestral N-terminal mitochondrial targeting sequence from the open reading frame. Guinea pig AGT is targeted to peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) peroxisomal import machinery, even though its C-terminal tripeptide, HRL, deviates from the standard consensus PTS1 motif. Although HRL appears to target AGT to peroxisomes less efficiently than the classical PTS1 SKL, the main reason for the low efficiency of AGT peroxisomal targeting in guinea pig cells (compared with cells from other species) lies not with guinea pig AGT but with some other, as yet undefined, part of the guinea pig peroxisomal import machinery.

Molecular characterization and clinical use of a polymorphic tandem repeat in an intron of the human alanine:glyoxylate aminotransferase gene.

The autosomal recessive disease primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate amino-transferase (AGT). This paper concerns the identification, characterization and clinical use of an unusual discretely polymorphic tandem repeat sequence in the fourth intron of the human AGT gene (gene locus designation AGXT). In a random Caucasian population, three alleles could be clearly recognized that consisted of either 12 (type III), 17 (type II) or approximately 38 (type I) tandemly repeated copies of a highly conserved 29/32-bp sequence with frequencies of 33%, 7% and 60%, respectively. In a random Japanese population, the allelic frequencies were markedly different (i.e. 31%, 45% and 19%, respectively). In addition, a fourth allele was identified, consisting of approximately 32 repeats (type IV), with an allelic frequency of approximately 5% in Japanese. The repetitive sequence was similar to previously identified mammalian sequences with homology to the Epstein-Barr virus IR3 repetitive element involving a 12/15-bp region GCA(GGN)GGAGGAGGG within the repeat unit. This IR3-like sequence was interspersed with a 17-bp sequence with no similarity to any currently known repetitive element. The type I and type III alleles were judged to be equivalent to a previously identified TaqI polymorphism. Two polymorphisms previously shown to be associated with the peroxisome-to-mitochondrion mistargeting of AGT in PH1 (a C154-->T point substitution in exon 1 and a 74-bp duplication in intron 1) were found to segregate exclusively with the type I intron 4 polymorphism in Caucasians, but not in Japanese. The polymorphic nature of the intron 4 tandem repeats makes them of potential use in the prenatal diagnosis of PH1, especially when coupled with the exon 1 C154-->T substitution or intron 1 duplication polymorphisms. A PH1 family, in which a fetus had been predicted previously to be either normal or a carrier by AGT enzymic analysis of a fetal liver biopsy, but who had been shown to be only partially informative with respect to the C154-->T/intron 1 polymorphisms, was analysed retrospectively. The family was completely informative for the intron 4 tandem repeat polymorphism and the carrier status of the fetus was confirmed.