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B-Myb regulates the A(2B) adenosine receptor in vascular smooth muscle cells.

ABSTRACT: The A(2B) adenosine receptor (A(2B)AR) has been described to control various vascular functions, including inhibition of smooth muscle cell proliferation. Here, we sought to understand the regulation of A(2B)AR gene expression in aortic vascular smooth muscle cells (VSMCs), with a focus on the proliferation phase. Assays with A(2B)AR-beta-gal aortic VSMCs, in which the endogenous A(2B)AR gene promoter drives the expression of prokaryotic beta-galactosidase (beta-gal) instead of the endogenous A(2B)AR gene, show that beta-gal expression is upregulated when the cells are induced to exit from cell cycle arrest. Similarly, the level of A(2B)AR mRNA is upregulated in proliferating primary aortic VSMCs. In search of related mechanisms, it was noted that the A(2B)AR gene promoter contains several putative binding sites for the proliferation-inducing transcription factor, B-Myb. Using a clone of the 5' region upstream of the mouse A(2B)AR gene linked to a reporter gene, B-Myb site deletion mutants were generated. It was determined that B-Myb upregulates the A(2B)AR gene promoter, and specific promoter binding sites were identified as functional. In accordance, B-Myb also elevates endogenous A(2B)AR mRNA and receptor activity, and this activity decreases cell proliferation. Our data are novel in that they show that this proliferation-inhibiting A(2B)AR is itself an inducible receptor regulated by B-Myb.

SUBMITTER: St Hilaire C

PROVIDER: S-EPMC3303598 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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B-Myb regulates the A(2B) adenosine receptor in vascular smooth muscle cells.

St Hilaire Cynthia C Yang Dan D Schreiber Barbara M BM Ravid Katya K

Journal of cellular biochemistry 20080401 6

The A(2B) adenosine receptor (A(2B)AR) has been described to control various vascular functions, including inhibition of smooth muscle cell proliferation. Here, we sought to understand the regulation of A(2B)AR gene expression in aortic vascular smooth muscle cells (VSMCs), with a focus on the proliferation phase. Assays with A(2B)AR-beta-gal aortic VSMCs, in which the endogenous A(2B)AR gene promoter drives the expression of prokaryotic beta-galactosidase (beta-gal) instead of the endogenous A( ...[more]

PMID: 17979185

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Project description:ObjectiveWe examined the role of syndecan-1 in modulating the phenotype of vascular smooth muscle cells in the context of endogenous inflammatory factors and altered microenvironments that occur in disease or injury-induced vascular remodeling.Methods and resultsVascular smooth muscle cells (vSMCs) display a continuum of phenotypes that can be altered during vascular remodeling. While the syndecans have emerged as powerful and complex regulators of cell function, their role in controlling vSMC phenotype is unknown. Here, we isolated vSMCs from wild type (WT) and syndecan-1 knockout (S1KO) mice. Gene expression and western blotting studies indicated decreased levels of α-smooth muscle actin (α-SMA), calponin, and other vSMC-specific differentiation markers in S1KO relative to WT cells. The spread area of the S1KO cells was found to be greater than WT cells, with a corresponding increase in focal adhesion formation, Src phosphorylation, and alterations in actin cytoskeletal arrangement. In addition, S1KO led to increased S6RP phosphorylation and decreased AKT and PKC-α phosphorylation. To examine whether these changes were present in vivo, isolated aortae from aged WT and S1KO mice were stained for calponin. Consistent with our in-vitro findings, the WT mice aortae stained higher for calponin relative to S1KO. When exposed to the inflammatory cytokine TNF-α, WT vSMCs had an 80% reduction in syndecan-1 expression. Further, with TNF-α, S1KO vSMCs produced increased pro-inflammatory cytokines relative to WT. Finally, inhibition of interactions between syndecan-1 and integrins αvβ3 and αvβ5 using the inhibitory peptide synstatin appeared to have similar effects on vSMCs as knocking out syndecan-1, with decreased expression of vSMC differentiation markers and increased expression of inflammatory cytokines, receptors, and osteopontin.ConclusionsTaken together, our results support that syndecan-1 promotes vSMC differentiation and quiescence. Thus, the presence of syndecan-1 would have a protective effect against vSMC dedifferentiation and this activity is linked to interactions with integrins αvβ3 and αvβ5.

Project description:c-Kit+ progenitor smooth muscle cells (P-SMCs) can develop into SMCs that contribute to injury-induced neointimal thickening. Here, we investigated whether adenosine reduces P-SMC migration and proliferation and whether this contributes to adenosine's inhibitory actions on neointima formation. In human P-SMCs, 2-chloroadenosine (stable adenosine analogue) and BAY60-6583 (A2B agonist) inhibited P-SMC proliferation and migration. Likewise, increasing endogenous adenosine by blocking adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl) adenine (inhibits adenosine deaminase) and 5-iodotubercidin (inhibits adenosine kinase) attenuated P-SMC proliferation and migration. Neither N6-cyclopentyladenosine (A1 agonist), CGS21680 (A2A agonist), nor N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (A3 agonist) affected P-SMC proliferation or migration. 2-Chloroadenosine increased cyclic AMP, reduced Akt phosphorylation (activates cyclin D expression), and reduced levels of cyclin D1 (promotes cell-cycle progression). Moreover, 2-chloroadenosine inhibited expression of Skp2 (promotes proteolysis of p27Kip1) and upregulated levels of p27Kip1 (negative cell-cycle regulator). A2B receptor knockdown prevented the effects of 2-chloroadenosine on cyclic AMP production and P-SMC proliferation and migration. Likewise, inhibition of adenylyl cyclase and protein kinase A rescued P-SMCs from the inhibitory effects of 2-chloroadenosine. The inhibitory effects of adenosine were similar in male and female P-SMCs. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 ?mol/L for 7 days) reduced neointimal hyperplasia by 64.5% (P<0.05; intima/media ratio: control, 1.4±0.02; treated, 0.53±0.012) and reduced neointimal c-Kit+ cells. Adenosine inhibits P-SMC migration and proliferation via the A2B receptor/cyclic AMP/protein kinase A axis, which reduces cyclin D1 expression and activity via inhibiting Akt phosphorylation and Skp2 expression and upregulating p27kip1 levels. Adenosine attenuates neointima formation in part by inhibiting infiltration and proliferation of c-Kit+ P-SMCs.

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B-Myb regulates the A(2B) adenosine receptor in vascular smooth muscle cells.

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B-Myb regulates the A(2B) adenosine receptor in vascular smooth muscle cells.

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