Transcriptional regulation of the CART promoter in CATH.a cells

Abstract

Changes in Cocaine- and Amphetamine-Regulated Transcript (CART) mRNA levels have been observed in brain as a result of various physiologic stimuli including feeding, drugs of abuse, stress and glucocorticoids, and activators of the cyclic AMP (cAMP) and protein kinase A (PKA) pathway. Accordingly, we are interested in identifying factors involved in CART gene regulation. CATH.a cells, derived from the locus coeruleus (LC), express a 213-bp CART mRNA species that is translated and processed. The promoter activity of three CART-LUC constructs containing 3451, 641, and 102 bp of 5′ upstream sequence, respectively, were tested in CATH.a cells. cAMP regulation was detected in the construct containing 641 bp of CART promoter sequence which contains a consensus CRE site. Mutation of the CRE site within −641CART-LUC significantly reduced basal and forskolin-induced promoter activity. Additionally, forskolin-induced transcription was inhibited by a dominant-negative mutant of CRE-binding protein (CREB) in CATH.a cells. Finally, tropin-releasing factor (CRF), an endogenously occurring activator of the cAMP/PKA pathway in CATH.a cells, was shown to increase transcriptional activity that was inhibited by a CRF receptor antagonist and a PKA inhibitor. This study provides evidence that the CRE site in the CART proximal promoter is involved in cAMP/PKA/CREB regulation in cells having a neuronal phenotype. Also, given the evidence for involvement of CREB in reward and reinforcement, these results are compatible with a role for CART in these processes as well.