Identification of Overlapping AP-2/NF-κB-responsive Elements on the Rat Cholecystokinin Gene Promoter

Abstract

In this study we evaluate both proximal and more distal transcriptional regulation of the 5 flanking region of the rat cholecystokinin gene in transfected GH3 (rat pituitary tumor) cells. Transcriptional activity was measured on the intact (؊400 to ؉73) 5 flanking region of cholecystokinin (CCK), as well as with DNA constructs, which were deleted in both the conventional 5 to 3, as well as an unconventional 3 to 5 direction. Our in vivo studies indicate complex phorbol ester and forskolin interactions in the 10-base pair region between ؊130 and ؊140. We conclude, there are at least two transcriptional factors involved in regulation of the rat CCK transcription in this region. In vitro studies utilizing heterologous nuclear (HeLa) extract, as well as purified transcription factors AP-2 and NF-B, identify overlapped AP-2-and NF-B-responsive elements within the 17-base pair sequence between ؊149 and ؊134 of the distal 5 flanking region. In this region complex transcriptional regulation occurs, which indicates inhibition of AP-2 CCK promoter complexing by NF-B. Sixpoint mutations introduced into this sequence prevent AP-2 and NF-B binding to CCK promoter, as well as its transcriptional activation by phorbol ester and forskolin in GH3 cells. Cholecystokinin (CCK),1 a prototypical brain-gut peptide (1-5), has hemacrine (3, 6) and autocrine (7) action in the gut. In the brain CCK is a neurotransmitter (8, 9). There is a commonly accepted gut, as well as brain, CCK transcription initiation site in the rat (10), mouse (11), and man (12). Alternatively, more distal 5Ј CCK transcription start sites have been identified in the brain of the rat (13). Transcriptional control of CCK is regulated by multiple factors. These include phorbol ester (14 -16), cAMP (14,(17)(18)(19)(20), and gonadal steroids (21), as well as glucocorticoids (22). Several cis-elements, important in transcriptional control, have been identified within the first 119 bp of the 5Ј flanking region of the rat CCK gene (10). This 119-bp fragment contains a sequence homologous with a 12-Otetradecanoylphorbol 13-acetate (TPA)-responsive element of the c-fos gene, as well as a cAMP-responsive element identified in the proenkephalin gene (23). This sequence binds several transcription factors including AP-1, jun, and fos homodimers, as well as cAMP-responsive element-binding protein (16,19). Two other recognition sites on the human CCK gene are Sp1 (at Ϫ39 to Ϫ34) and E-box or upstream stimulatory factor (at Ϫ97 to Ϫ92) (16). Additionally, a negative interaction was detected between TPA-responsive element and E-box or upstream stimulatory factor-responsive element (24).TPA and forskolin activate specific nuclear proteins, which regulate gene transcription. These proteins include AP-2 (25) and NF-B (26, 27). Transcription factor AP-2 is activated either directly or indirectly by two signal transduction pathways (25). One route involves cAMP-dependent protein kinase A. The other involves phorbol ester and diacylglycerol-activated protein kinase C (28). AP-2 specifi...

NF-κB inhibits transcription of the H+-K+-ATPase α2-subunit gene: role of histone deacetylases

“…The transcription factors examined were NF-B p50 and, as a control, AP-2. For the NF-B experiment, the same final binding buffer was used as described (21). Briefly, the labeled template DNA was incubated in a final volume of 50 l with or without 2 l of recombinant human NF-B in the final buffer containing 10 mM HEPES (pH 7.9), 0.2 mM EDTA, 50 mM KCl, 2.5 mM dithiothreitol, 10% glycerol, and 0.05% Nonidet P-40 for 10 min on ice.…”

AP2α alters the transcriptional activity and stability of p53

“…AP2 and p53 act synergistically to induce expression of the gelatinase A (matrix metalloproteinase 2) gene promoter (Mertens et al, 2002) and the KAI1 gene promoter (Marreiros et al, 2005). In hepatocytes, AP2 factors act in a coordinate fashion with NF-kB to regulate the cholecystokinin promoter (Katsel and Greenstein, 2001) and the serum amyloid A1 (SAA) promoter (Ren and Liao, 2001). AP2 has been reported to act synergistically with retinoic acid receptors to activate the CRABPII (Astrom et al, 1992;Oulad-Abdelghani et al, 1996) and the HOX A4 gene promoters (Doerksen et al, 1996).…”

PPARα and AP-2α regulate bombesin receptor subtype 3 expression in ozone-stressed bronchial epithelial cells

“…The expression of AP-2α is associated with the embryonic differentiation and cancer [27,28]. Also, AP-2α has been shown to regulate neuropeptide receptors [29,30] and neuropeptide genes [31,32]. However, the activation and translocation of AP-2α remained unclear.…”