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Role of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production.

ABSTRACT: Interstitial adenosine stimulates neovascularization in part through A2B adenosine receptor-dependent upregulation of vascular endothelial growth factor (VEGF). In the current study, we tested the hypothesis that A2B receptors upregulate JunB, which can contribute to stimulation of VEGF production. Using the human microvascular endothelial cell line, human mast cell line, mouse cardiac Sca1-positive stromal cells, and mouse Lewis lung carcinoma (LLC) cells, we found that adenosine receptor-dependent upregulation of VEGF production was associated with an increase in VEGF transcription, activator protein-1 (AP-1) activity, and JunB accumulation in all cells investigated. Furthermore, the expression of JunB, but not the expression of other genes encoding transcription factors from the Jun family, was specifically upregulated. In LLC cells expressing A2A and A2B receptor transcripts, only the nonselective adenosine agonist NECA (5'-N-ethylcarboxamidoadenosine), but not the selective A2A receptor agonist CGS21680 [2-p-(2-carboxyethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine], significantly increased JunB reporter activity and JunB nuclear accumulation, which were inhibited by the A2B receptor antagonist PSB603 [(8-[4-[4-((4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine]. Using activators and inhibitors of intracellular signaling, we demonstrated that A2B receptor-dependent accumulation of JunB protein and VEGF secretion share common intracellular pathways. NECA enhanced JunB binding to the murine VEGF promoter, whereas mutation of the high-affinity AP-1 site (-1093 to -1086) resulted in a loss of NECA-dependent VEGF reporter activity. Finally, NECA-dependent VEGF secretion and reporter activity were inhibited by the expression of a dominant negative JunB or by JunB knockdown. Thus, our data suggest an important role of the A2B receptor-dependent upregulation of JunB in VEGF production and possibly other AP-1-regulated events.

SUBMITTER: Ryzhov S

PROVIDER: S-EPMC3868903 | biostudies-other | 2014 Jan

REPOSITORIES: biostudies-other

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Role of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production.

Ryzhov Sergey S Biktasova Asel A Goldstein Anna E AE Zhang Qinkun Q Biaggioni Italo I Dikov Mikhail M MM Feoktistov Igor I

Molecular pharmacology 20131017 1

Interstitial adenosine stimulates neovascularization in part through A2B adenosine receptor-dependent upregulation of vascular endothelial growth factor (VEGF). In the current study, we tested the hypothesis that A2B receptors upregulate JunB, which can contribute to stimulation of VEGF production. Using the human microvascular endothelial cell line, human mast cell line, mouse cardiac Sca1-positive stromal cells, and mouse Lewis lung carcinoma (LLC) cells, we found that adenosine receptor-depen ...[more]

PMID: 24136993

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Project description:BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A(2B) receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A(2B) receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-?B. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A?and?CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

Project description:Fabry disease is a lysosomal storage disorder (LSD) caused by deficiency of α-galactosidase A (α-gal A), resulting in deposition of globotriaosylceramide (Gb3; also known as ceramide trihexoside) in the vascular endothelium of many organs. A gradual accumulation of Gb3 leads to cardiovascular, cerebrovascular and renal dysfunction. Endothelial cell dysfunction leads to renal complications, one of the main symptoms of Fabry disease. However, the pathological mechanisms by which endothelial dysfunction occurs in Fabry disease are poorly characterized. The purpose of this study was to investigate whether the expression of transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) is associated with the renal pathogenesis of Fabry disease. We found that the protein expression levels of renal thrombospondin-1 (TSP-1), TGF-β1 and VEGF were higher in the kidneys from Fabry mice compared to wild-type mice. The expression levels of VEGF receptor 2 (VEGFR2), fibroblast growth factor-2 (FGF-2) and phospho-p38 (P-p38) were also higher in the kidneys from Fabry mice compared with wild-type mice. Activities of cysteine aspartic acid protease (caspase)-6 and caspase-9 were higher in kidneys from Fabry than from the wild-type mice. These results suggest that overexpression of TGF-β1 and VEGF in the Fabry mouse kidney might contribute to Fabry disease nephropathy by inducing apoptosis. To test whether Gb3 accumulation can induce apoptosis, we incubated bovine aortic endothelial cells with Gb3 and found increased expression of TGF-β1, VEGFR2, VEGF, FGF-2 and P-p38. The combination of increased expression of TGF-β1 and VEGF caused by Gb3 accumulation may allow upregulation of FGF-2, VEGFR2 and P-p38 expression, and these changes may be associated with Fabry disease nephropathy by inducing apoptosis.

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Role of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production.

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Role of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production.

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