AP-1 is involved in UTP-induced osteopontin expression in arterial smooth muscle cells.

Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The -96 to +1 region mediated UTP-induced OPN overexpression (+276+/-60%). Sequence analysis of this region revealed a potential site for AP-1 located at -76. When this AP-1 site was deleted, UTP-induced activation of the -96 to +1 region was totally inhibited. Thus, this AP-1 (-76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation.

[Mechanisms of regulation of osteopontin expression mediated by UTP, angiotensin II and PDGF in arterial smooth muscle cells]. / Mécanismes de régulation de l'expression de l'ostéopontine par l'UTP, l'angiotensine II et le PDGF dans les cellules musculaires lisses artérielles.

Osteopontin (OPN), an RGD containing extracellular matrix protein, is associated with arterial smooth muscle cells (SMC) activation in vitro and in vivo. OPN has been shown to be overexpressed in vascular injury. Its expression can be induced by many factors including growth factors, cytokines, hormones and extracellular nucleotides. We are interested in understanding mechanisms regulating the OPN mRNA steady state level in SMC. We compared the effect of two G-protein coupled receptors agonists (UTP and angiotensin II [AII]) and one tyrosin kinase receptor agonist (PDGF). We explored the effect of these three agonists both on OPN transcription using gene reporter assay and on OPN mRNA stabilisation using actinomycin D. We showed that UTP 100 microM. AII 10 microM and PDGF 50 ng/microL induced OPN transcription. Whereas UTP and AII induced a 366 +/- 81% and 338 +/- 115% activation of transcription respectively, PDGF demonstrated a lower efficiency (195 +/- 59%) inducing the transcription. Moreover, we demonstrated that UTP and AII but not PDGF were able to stabilize OPN mRNA. This effect seems to be specific to G-protein coupled receptor agonists since previous studies demonstrated that intracellular receptor agonists did not stabilise OPN mRNA. Thus, the lower increase of OPN mRNA level in response to PDGF stimulation compared to AII or UTP could be explain by both, the lower activation of the OPN promoter and the effect of UTP and AII on OPN mRNA stabilisation.

YLR209c encodes Saccharomyces cerevisiae purine nucleoside phosphorylase.

The yeast YLR209c (PNP1) gene encodes a protein highly similar to purine nucleoside phosphorylases. This protein specifically metabolized inosine and guanosine. Disruption of PNP1 led to inosine and guanosine excretion in the medium, thus showing that PNP1 plays an important role in the metabolism of these purine nucleosides in vivo.

Role of adenosine kinase in Saccharomyces cerevisiae: identification of the ADO1 gene and study of the mutant phenotypes.

Sequencing of the Saccharomyces cerevisiae genome revealed an open reading frame (YJR105w) encoding a putative protein highly similar to adenosine kinases from other species. Disruption of this gene (renamed ADO1) affected utilization of S-adenosyl methionine (AdoMet) as a purine source and resulted in a severe reduction of adenosine kinase activity in crude extracts. Furthermore, knock-out of ADO1 led to adenosine excretion in the medium and resistance to the toxic adenosine analogue cordycepin. From these data we conclude that ADO1 encodes yeast adenosine kinase. We also show that ADO1 does not play a major role in adenine utilization in yeast and we propose that the physiological role of adenosine kinase in S. cerevisiae could primarily be to recycle adenosine produced by the methyl cycle.

Extracellular adenosine induces apoptosis of human arterial smooth muscle cells via A(2b)-purinoceptor.

Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A(1)/A(2)-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P(1) purinoceptor subtypes, only alloxazine, an antagonist of A(1)- and A(2)-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate, which are A(1)-, A(2a)-, and A(3)-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A(2b)-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A(2b)-adenosine receptor and involves a cAMP-dependent pathway.

Deoxyribosyl exchange reactions leading to the in vivo generation and regeneration of the antiviral agents (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine and 5-(2-chloroethyl)-2'-deoxyuridine.

In the rat, the highly potent anti-herpes drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd) is rapidly converted to its base (E)-5-(2-bromovinyl)uracil (BVUra) through the action of pyrimidine nucleoside phosphorylases. However, BVdUrd can be regenerated or even generated de novo from BVUra by a pentosyl transfer reaction upon the administration of 2'-deoxythymidine (dThd), 2'-deoxyuridine (dUrd) or 5-ethyl-2'-deoxyuridine (EtdUrd). The antiherpetic drugs EtdUrd and 5-(2-chloroethyl)-2'-deoxyuridine (ClEtdUrd) can also be regenerated or generated de novo from their respective bases 5-ethyluracil (EtUra) and 5-(2-chloroethyl)uracil (ClEtUra), by a pentosyl transfer mediated by the administration of dThd or dUrd as deoxyribosyl donor. The generation or regeneration of BVdUrd, EtdUrd and ClEtdUrd from their bases (BVUra, EtUra and ClEtUra, respectively) is readily achieved because the latter have long half-lifes. Thus, the active anti-herpes drugs can be (re)generated repeatedly after a single administration of these nucleosides or their bases, followed by repeated administrations of dUrd.