Proximal promoter of the rat brain creatine kinase gene lacks a consensus CRE element but is essential for the cAMP-mediated increased transcription in glioblastoma cells

• Published in: Journal of neuroscience research 1999-05, Vol.56 (4), p.371-385
• Main Authors: Kuzhikandathil, Eldo V., Molloy, George R.
• Format: Article
• Language: English
• Subjects: 1-Methyl-3-isobutylxanthine - pharmacology; Animals; Astrocytes - drug effects; Astrocytes - enzymology; Astrocytes - metabolism; Base Sequence; Brain - enzymology; Colforsin - pharmacology; Consensus Sequence - genetics; Creatine Kinase - genetics; cyclic AMP; Cyclic AMP - pharmacology; differentiation; DNA Probes; energy metabolism; Gene Expression Regulation, Enzymologic - drug effects; glial cells; Glioblastoma; Humans; Introns - genetics; Nuclear Proteins - metabolism; Promoter Regions, Genetic - genetics; Rats; Response Elements - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; TATA Box - genetics; transcription; Transcriptional Activation - drug effects; Transfection; Tumor Cells, Cultured
• ISSN: 0360-4012; 1097-4547
• DOI: 10.1002/(SICI)1097-4547(19990515)56:4<371::AID-JNR5>3.0.CO;2-W

Our previous studies have shown that transcription of brain creatine kinase (CKB) mRNA in U87‐MG glioblastoma cells is stimulated by a forskolin‐mediated increase in cyclic AMP (cAMP) via a pathway involving protein kinase A (PKA) and the activation of Gαs proteins. In this report, we have employed transient transfection to investigate the rat CKB gene elements essential for the cAMP‐mediated induction of rat CKB transcription in human U87 cells and have mapped the transcription start site of the induced CKB transcripts. We found that the level of induced transcription from the transfected genomic rat CKB gene was the same whether transcription was driven by 2.9 kb of CKB promoter plus 5′ flanking sequence or the 0.2 kb CKB promoter, suggesting that the proximal CKB promoter was essential. Also, the level of induced transcription of the chloramphenicol acetyl transferase (CAT) reporter gene driven by the 2.9 kb CKB promoter was the same as with the 0.2 kb CKB promoter. Analyses of a series of 5′ deletions of the 0.2 kb proximal CKB promoter showed that the sequences between −80 bp and +1 bp were essential for the cAMP‐mediated induction of CKB transcription, despite the absence of a consensus cAMP response element (CRE) sequence in that region. In agreement, gel mobility shift assays showed that nuclear extracts from U87 cells contained a protein(s) which bound specifically to a [32P]CKB DNA probe containing the −60 bp to +1 bp sequence. Mapping the 5′ end of the CKB transcripts showed that the initiation of the cAMP‐induced transcription occurred almost exclusively from the downstream transcription start site, apparently under the initiation direction of the nonconsensus (−28) TTAA element and not the consensus (−60) TATAAATA element. The results are discussed with regard to nuclear protein factors which may be involved, and the possible cAMP‐mediated increase in CKB transcription during myelinogenesis, since the differentiation of oligodendrocytes has previously been shown to be accelerated by increased intracellular cAMP. J. Neurosci. Res. 56:371–385, 1999. © 1999 Wiley‐Liss, Inc.