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.

Molecular "rehabilitation" by rational drug targeting: the challenge of P53 in cancer treatment.

The p53 tumor suppressor protein is a pivotal molecule in the protection against carcinogenesis and is inactivated in a plethora of human neoplasms, p53 prevents the accumulation of genomic alterations by inducing cell-cycle arrest or programmed cell death in response to genotoxic signals such as DNA damage and stress. In as much as p53 is a key modulator of cell proliferation, tight regulation of its function is critical for normal growth and homeostasis of cells and tissues. This is achieved by various mechanisms including the ability of the protein to adopt active and latent forms, its cellular localization, and control of p53 stability. The vast array of data on the atomic structure of the p53 protein, in conjunction with recent insights into the biochemical mechanisms that fine-tune its activity open up exciting new avenues for the development of rational therapeutic approaches aiming at restoring p53 function in a broad spectrum of human malignancies.