CREB CONTROLS LAP C EBP-BETA TRANSCRIPTION

LAP/C/EBP beta is a member of the C/EBP family of transcription factor s and is involved in hepatocyte-specific gene expression, Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP bet a mRNA is modulated during liver regeneration. Therefore, in this stud y we investigated mechanisms which control LAP/C/EBP beta gene transcr iption, Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonst rated that a small region in close proximity to the TATA box is import ant in maintaining a high level of transcription of the luciferase rep orter gene constructs. In gel shift experiments two sites were identif ied which are important for specific complex formation within this reg ion. Further analysis by cross-linking, super shift, and competition e xperiments was performed with liver cell nuclear extracts, hepatoma ce ll nuclear extracts, or recombinant CREB protein, These experiments co nclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensu s sequence. Transfection experiments with promoter constructs where th e CREB sites were mutated showed that these sites are important to mai ntain both basal promoter activity and LAP/C/EBP beta inducibility thr ough CREB. Northern blot analysis and runoff transcription assays demo nstrated that the protein kinase A pathway not only stimulated the act ivity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types, Western blot analysis of rat liver cell nuclear extracts and runoff transcrip tion assays of rat liver cell nuclei after two-thirds hepatectomy show ed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These re sults demonstrate that the two CREB sites are important to control LAP /C/EBP beta transcription in vivo, As several pathways control CREB ph osphorylation, our results provide evidence for the transcriptional re gulation of LAP/C/EBP beta via CREB under different physiological cond itions.