Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.

Abstract

We have examined the regulation of somatostatin gene expression by cAMP in PC12 rat pheochromocytoma cells transfected with the rat somatostatin gene. Forskolin at 10 jIM caused a 4-fold increase in somatostatin mRNA levels within 4 hr of treatment in stably transfected cells. Chimeric genes containing the somatostatin gene promoter fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase were also induced by cAMP in PC12 cells. To delineate the sequences required for response to cAMP, we constructed a series of promoter deletion mutants. Our studies defined a region between 60 and 29 base pairs upstream from the transcriptional initiation site that conferred cAMP responsiveness when placed adjacent to the simian virus 40 promoter. Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP. cAMP responsiveness was greatly reduced when the somatostatin fusion genes were transfected into the mutant PC12 line A126-1B2, which is deficient in cAMPdependent protein kinase 2. Our studies indicate that transcriptional regulation of the somatostatin gene by cAMP requires protein kinase 2 activity and may depend upon a highly conserved promoter element.

Mechanisms of action of glucagon-like peptide 1 in the pancreas

“…The cAMP response element (CRE, TGACGTCA) was initially recognized as an inducible enhancer of genes that can be transcribed in response to elevated cAMP levels (Comb et al, 1986;Montminy et al, 1986). This regulatory element has been characterized as being responsive to a number of basic region leucine zipper transcription (bZIP) factors; however the most studied is the cAMP response element binding (CREB) protein.…”

The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma

“…pD(À71)SomCAT contains the somatostatin promoter to position À71, fused to the chloramphenicol acetyl transferase (CAT) coding sequences (Montminy et al, 1986). pD(À42)SomCAT was obtained by digestion of pD(À71)SomCAT with Aat2, blunting with T4 DNA polymerase and religation (Brown et al, 1995).…”

Down‐regulation of the protein kinase A pathway by activators of protein kinase C and intracellular Ca2+ in fibroblast cells

“…To study the antagonism of the PKA, PKC and Ca 2+ pathways in mouse fibroblasts we used defined reporter plasmids containing cAMP responsive elements (CRE) or TPA responsive elements (TRE) in front of the chloramphenicol acetyl transferase (CAT) reporter gene. The CREs are targets of the transcription factor CREB [2] which is activated through the PKA and Ca2+/calmodulin pathways, whereas the TREs are targets of the products of the fos and jun gene families which are activated by the PKC pathway [3] and elevated levels of intracellular Ca 2+ [4]. The results we obtained with this system show that the PKC and Ca 2+ pathways dominate over the PKA pathway in mouse fibroblasts when they are stimulated at the same time.…”

“…Chloramphenicol acetyl transferase (CAT) assays were performed mainly following the protocol of Gorman et al [11] with the exception that the whole-cell extracts were heated to 65°C for 10 rain to destroy deacylases and that the incubation time was extended to 16 h. As reporter genes we used 2×SOM-CRE-CAT [12] containing two CREs of the somatostatin gene [2] in front of a TATA-box and the CAT gene; and 5× TRE-CAT [13] in which five TPEs [3] have been inserted in front of a TATA-box and the CAT gene. For each experiment three independent transfections were carried out and each whole-cell extract obtained was tested twice for CAT activity.…”