CELL-SPECIFIC EXPRESSION OF THE RAT SECRETOGRANIN-II PROMOTER

Secretogranin II (SgII) is a member of the granin family of secretory proteins, which are selectively expressed in neuroendocrine cells. As a first step in understanding the molecular basis for cell type-specif ic expression of SgII, we isolated a 12-kb clone from a rat genomic li brary that contained the entire rat SgII coding region, the transcript ion initiation site, and approximately 3 kb of 5'-flanking region. Wit hin 75 bp of the transcription start site (+1) we located a TATA box a nd a consensus cAMP responsive element. Within the 5'-flanking region, a number of potential cis-acting elements were identified, including 2 Pit-1 binding sites, 15 E box motifs, and near-perfect matches for A P-1 and AP-2 sites. To demonstrate cell type-specific expression the r at SgII gene, a plasmid containing 2.6 kb of the 5'-flanking region of the SgII gene fused to the luciferase reporter gene (p2774Luc) was tr ansfected into rat pheochromocytoma PC-12 cells, rat pituitary GH(4)C( 1) (GH) cells, human BE(2)-M17 (M17) neuroblastoma cells, and mouse fi broblast NIH/3T3 cells. The promoter activity was 6- to 36-fold higher in neuroendocrine cells than in NIH/3T3 cells. Progressive deletions in the 5'-flanking region to 61 bp upstream of the start site (p223Luc ) had no effect on promoter activity in PC-12 cells. On the other hand , a 5'-deletion in the SgII promoter to -1032 increased promoter activ ity 3.8-fold in GH cells. This level of expression was maintained when the SgII promoter was further truncated to -189, whereas truncation t o -61 resulted in a 2.6-fold reduction in promoter activity. These res ults suggest that the sequence between -61 and +162 bp is sufficient f or SgII promoter activity in PC-12 cells. However, other elements in t he 5'-flanking region contribute to both positive and negative regulat ion of the rat SgII gene in GH cells.