Regulation of the preprotachykinin-I gene promoter through a protein kinase A-dependent, cyclic AMP response element-binding protein-independent mechanism.
Preprotachykinin-I (PPT) gene expression is regulated by a number of stimuli that signal through cyclic AMP (cAMP)-mediated pathways. In the present study, forskolin, an adenylyl cyclase stimulator, significantly increased PPT mRNA levels in PPT-expressing RINm5F cells, an effect paralleled by an increase in PPT promoter-luciferase reporter construct activity. The forskolin-induced stimulation of PPT transcription was protein kinase A dependent (PKA), as shown by blockade with the PKA inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide. We found that the activation protein 1/cAMP response element (AP1/CRE) site centered at -196 relative to the transcription start site was important for basal and forskolin-induced PPT promoter activity. Because of the involvement of PKA and the similarity of the AP1/CRE element to consensus CRE sequences, we investigated the role of CRE-binding protein (CREB) in the regulation of the PPT promoter. Surprisingly, overexpression of a dominant-negative CREB (i.e. CREB-A) did not affect basal or forskolin-induced PPT promoter activity. Furthermore, binding of CREB to the PPT promoter AP1/CRE site was not demonstrable in electrophoretic mobility shift assays. Rather, our experiments suggested that c-Jun is a member of the complex that binds to this site. We conclude that, at least in RINm5F cells, cAMP-mediated up-regulation of PPT gene expression does not involve CREB or CREB-related transcription factor recruitment to the AP1/CRE site.
Calin-Jageman
Irina E
IE
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Wang
Jun
J
Bannon
Michael J
MJ
D000818:Animals; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019143:Evolution, Molecular; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000502:physiology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D015320:Tachykinins / Q000096:biosynthesis / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D011498:Protein Precursors; D012333:RNA, Messenger; D015320:Tachykinins; D018808:Transcription Factor AP-1; C044206:preprotachykinin; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases