Characterization of the cocaine‐ and amphetamine‐regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP‐dependent signaling pathway in GH3 cells

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Abstract
Cocaine‐ and amphetamine‐regulated transcript (CART) peptides are regulated neuropeptides that play a role in a variety of physiological processes. CART mRNA is also highly regulated as its levels change in response to psychostimulant drugs and leptin. To understand the mechanisms involved in regulating CART mRNA levels, the mouse CART 5′‐flanking regulatory region was studied. The sequence of 3.4 kb of the mouse CART 5′‐flanking region revealed a proximal promoter that contains a cluster of transcription factor binding sites, including an overlapping STAT/CRE/AP1 site. In addition, the 5′‐most 320 bp of the CART promoter shares 83% nucleotide identity between mouse and human. Three luciferase expressing constructs containing varying amounts of CART 5′ upstream sequence were generated and tested for promoter activity. Transient transfection of GH3 cells with constructs containing 641 and 3451 bp of upstream sequence displayed strong promoter activity, producing 29‐fold and 51‐fold stimulation, respectively, while, a construct containing 102 bp of upstream sequence displayed a 5.4‐fold increase in activity. A␣construct containing the composite STAT/CRE/AP1 site was responsive to cyclic AMP induction by forskolin in GH3 cells. Forskolin treatment also resulted in a 4.5‐fold increase in CART mRNA levels after 6 h and the addition of H89, an inhibitor of protein kinase A, reduced the levels by 50%. These studies indicate that the CART proximal promoter lies within the 5′‐most 641 bp and that in GH3 cells the CART gene is regulated via a cyclic AMP‐dependent pathway.