Adenosine A3 receptor-induced proliferation of primary human coronary smooth muscle cells involving the induction of early growth response genes

Abstract In human coronary smooth muscle cells adenosine A 2B receptors mediate the inhibition of platelet-derived growth factor (PDGF)-induced proliferation via induction of the transcription factor nuclear receptor subfamily 4, group A, member 1 (NR4A1). In the absence of PDGF, adenosine analogues increased proliferation. In the present study we characterised the adenosine receptor mediating the increase in proliferation of these cells and identified involved transcription factors. Cultured human coronary smooth muscle cells were treated with selective A 3 receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The A 3 receptor agonist 2-chloro-IB-MECA (2-chloro-N 6 -(3-iodobenzyl)adenosine-5′-N-methylcarboxamide) enhanced the number of human coronary smooth muscle cells with a half-maximal concentration of only 1 nM. 2-chloro-IB-MECA also increased the expression of the transcription factors early growth response protein (EGR)2 and EGR3, but not of EGR1, NR4A1, NR4A2 and NR4A3. The responses to 2-chloro-IB-MECA were blocked by two A 3 receptor antagonists, MRS1523 (3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate; 10–300 μM) and VUF 5574 (N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea; 1–100 nM, as well as by the phospholipase C-inhibitor U73343 (0.2 μM). Small interfering RNA directed against EGR2 and EGR3 abolished the increases in proliferation induced by 2-chloro-IB-MECA. In summary, this is the first report demonstrating a coupling of smooth muscle adenosine A 3 receptors to increases in proliferation of human coronary smooth cells by the activation of phospholipase C and an induction of the transcriptions factors EGR2 and EGR3. The results facilitate the understanding of the role of adenosine A 3 receptors in the cardiovascular system.