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
✍ Scribed by Eldo V. Kuzhikandathil; George R. Molloy
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 304 KB
- Volume
- 56
- Category
- Article
- ISSN
- 0360-4012
No coin nor oath required. For personal study only.
✦ Synopsis
Our previous studies have shown that transcription of brain creatine kinase (CKB) mRNA in U87-MG glioblastoma cells is stimulated by a forskolinmediated increase in cyclic AMP (cAMP) via a pathway involving protein kinase A (PKA) and the activation of G␣ s 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 5Ј 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 5Ј 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 [ 32 P]CKB DNA probe containing the ؊60 bp to ؉1 bp sequence. Mapping the 5Ј 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 acceler-ated by increased intracellular cAMP.