Regulation of the A3 adenosine receptor gene in vascular smooth muscle cells: role of a cAMP and GATA element.

In previous studies, we reported that the level of expression of the adenylyl cyclase inhibitory A3 adenosine receptor (AR) impacts vascular tone and that rat vascular smooth muscle cells (VSMCs) coexpress the A3 AR and the adenylyl cyclase stimulatory A2a- and A2b-type ARs. In the current study, we investigated the regulation of expression of the A3 AR gene, focusing on sequences conserved in the mouse and human promoters. Transient transfection of primary cultures of rat VSMCs, using the mouse A3 AR promoter, shows that mutation of a conserved cAMP response element (CRE) significantly up-regulates promoter activity in first passage cells, whereas mutation of a conserved GATA site reduces promoter activity. This suggests that an inhibitory protein binds the CRE, whereas an enhancing factor binds the GATA sequence. Electrophoretic mobility shift assays (EMSAs) indicate that the putative CRE and GATA sites indeed bind cAMP response element modulator 1/c-Jun and the GATA6 protein, respectively. A3 AR promoter activity is significantly up-regulated in the presence of forskolin, the nonselective agonist 5'-(N-ethylcarboxamido)adenosine, or the A2a AR agonist 4-[2-[[6-amino-9(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepro- panoic acid (CGS21680), reaching levels similar to those of the A3 AR promoter bearing a mutated CRE. EMSA indicates that in the presence of forskolin the binding to the CRE is inhibited, suggesting that cAMP elevation disturbs the formation of an inhibitory complex on the CRE. Finally, semiquantitative reverse transcription-polymerase chain reaction analysis reveals that endogenous A3 AR mRNA is elevated in response to forskolin. Our findings suggest the presence of a mechanism by which cAMP might control its own level in cells via regulation of genes involved in modulation of adenylyl cyclase activity.

Rat NAP1: cDNA cloning and upregulation by Mpl ligand.

The Mpl ligand is a hematopoietic cytokine which exerts its effects through association with the c-Mpl receptor. It regulates the proliferation, polyploidization and maturation of platelet precursors, the megakaryocytes. Using a differential display polymerase chain reaction (PCR) approach, we have identified an mRNA, belonging to a family of nucleosome assembly proteins, whose expression is upregulated in response to Mpl ligand. Multiple size classes of this mRNA (1.7, 2.5 and 4.3kb) are readily detected in rat primary bone marrow cells and hematopoietic tissues. The size classes are also expressed to different extents in cell lines of all hematopoietic lineages. We isolated the full-length cDNA encoding the rat megakaryocyte 1.7kb mRNA, referred to as rNAP1. Bacterially expressed recombinant protein encoded by the 1.7kb cDNA facilitates the formation of nucleosomes on relaxed circular DNA in vitro. Our data indicate that rNAPs, which may facilitate chromatin reorganization, are upregulated by Mpl ligand. It is possible that NAPs contribute to Mpl ligand's induced effects on hematopoietic cells.