Gastric chief cell‐specific transcription of the pepsinogen A gene

Abstract

The molecular mechanisms underlying the regulation of pepsinogen A (PGA) gene expression in mammalian cells are poorly understood. In this paper we describe the structural and functional analysis of the pepsinogen A gene promoter in the pig. By genomic Southern analyses we demonstrate that, in contrast with human PGA genes which are amplified and organized in haplotypes, only a single PGA gene is present per haploid porcine genome. With the aim of identifying promoter elements mediating the gastric mucosa cell-specific transcription of the PGA gene in pig, we isolated a PGA gene from a porcine genomic library. The nucleotide sequence of the first exon and 1.7 kb of the upstream DNA region were determined and compared with the corresponding regions of the human PGA gene encoding isozymogen Pg5. In order to study the promoter activity 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 noncoding first exon sequences of the porcine and human PGA genes were linked to the chloramphenicol acetyltransferase (CAT) gene. The transcriptional activity of these hybrid genes was assessed in transient expression assays upon transfection (lipofection) of gastric and nongastric cells. 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 cell-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 the PGA gene. In contrast, all human PGA-CAT constructs used failed to show promoter activity in porcine gastric chief cells, indicating species-specific control of PGA gene expression. In addition, the transcriptional activity of the porcine PGA promoter in chief cells from pig was completely abolished by in vitro CpG methylation. Footprint analyses of the proximal promoter fragment using nuclear extracts from either porcine gastric mucosal chief cells or liver revealed some notable differences between both extracts, which might reflect the interaction with (a) cell-specific factor(s).

Identification and expression characterization of pepsinogen A in orange‐spotted grouper, Epinephelus coioides

“…In general, steady‐state pepsinogen A mRNA levels are only detected in gastric fundic mucosa, e.g. in human (Varis, 1985), pig (Meijerink et al , 1993), red porgy Pagrus pagrus (L.) (Darias et al , 2005) and Atlantic halibut Hippoglossus hippoglossus (L.) (Murray et al , 2006). However, pepsinogen A is also specifically expressed in the gill and skin of stomachless pufferfish T. rubripes (Kurokawa et al , 2005).…”

Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development

“…In the chicken prochymosin gene, GATA-and Sox-binding sequences have been found in the 5¢-flanking region and shown to be essential for stomach-specific expression, with binding of cGATA-5 protein causing upregulation [121]. The 5¢-flanking region of the gene for porcine pepsinogen A has been isolated, and the essential region for promotor activity identified [122]. Several sites from -246 to +21 act synergistically in transcriptional regulation of the gene.…”

Regulation of human and mouse procathepsin E gene expression

“…pronapsin A in human lung and kidney [1], prorenin in the juxtaglomerular cells of the kidney and in human reproductive organs [2], and progastricsin which is present in human stomach and is also secreted from the acinar cells lining the prostate so that the mature enzyme, gastricsin, is found in high concentrations in seminal fluid [3,4]. In contrast, pepsinogen A is produced only within the chief cells lining the human stomach [5]. Human gastric juice thus contains pepsin A and gastricsin.…”

“…Instead, both sequences contain an initiator element [14] which offers an alternative binding site for TFIID to initiate gene transcription [15]. While the factors that influence production of pepsinogen A, progastricsin, prorenin and procathepsin D have been studied [5,16–18], nothing is known about how the expression of the procathepsin E gene is regulated and how this can be correlated with the production of this intracellular aspartic proteinase only in certain cells. In the present report, we demonstrate that, whereas the extent of gene methylation does not affect transcription, expression of the procathepsin E gene is dependent upon the balance between the effects produced by positive‐acting, tissue‐specific transcription factors such as GATA1 and PU1 and the negative influence of the ubiquitous factor YY1.…”