GASTRIC CHIEF CELL-SPECIFIC TRANSCRIPTION OF THE PEPSINOGEN-A GENE

The molecular mechanisms underlying the regulation of pepsinogen A (PG A) gene expression in mammalian cells are poorly understood. In this p aper we describe the structural and functional analysis of the pepsino gen A gene promoter in the pig. By genomic Southern analyses we demons trate that, in contrast with human PGA genes which are amplified and o rganized in haplotypes, only a single PGA gene is present per haploid porcine genome. With the aim of identifying promoter elements mediatin g the gastric mucosa cell-specific transcription of the PGA gene in pi g, we isolated a PGA gene from a porcine genomic library. The nucleoti de sequence of the first exon and 1.7 kb of the upstream DNA region we re determined and compared with the corresponding regions of the human PGA gene encoding isozymogen Pg5. In order to study the promoter acti vity of the PGA gene a functional assay was developed: we succeeded in obtaining primary monolayer cultures of porcine gastric mucosal chief cells, suitable for transfection. Fragments of 5'-flanking and noncod ing first exon sequences of the porcine and human PGA genes were linke d to the chloramphenicol acetyltransferase (CAT) gene. The transcripti onal activity of these hybrid genes was assessed in transient expressi on assays upon transfection (lipofection) of gastric and nongastric ce lls. Whereas PGA 5'-flanking sequences showed no promoter activity in nongastric cell types, the DNA region from -205 to +21 was found to be sufficient to direct expression of the porcine PGA constructs in a ce ll-specific manner. Further deletion analysis of the proximal promoter fragment identified several regions (-205 to -167, -127 to -67 and +2 to +21) acting synergistically in the transcriptional regulation of t he PGA gene. In contrast, all human PGA-CAT constructs used failed to show promoter activity in porcine gastric chief cells, indicating spec ies-specific control of PGA gene expression. In addition, the transcri ptional activity of the porcine PGA promoter in chief cells from pig w as completely abolished by in vitro CpG methylation. Footprint analyse s of the proximal promoter fragment using nuclear extracts from either porcine gastric mucosal chief cells or liver revealed some notable di fferences between both extracts, which might reflect the interaction w ith (a) cell-specific factor(s).