We have previously reported that the zinc transporter hZTL1 is regulated at the mRNA level by zinc in human intestinal Caco-2 cells but not in the placental cell-line JAR (Cragg et al. 2002). Comparison of the cDNA sequences of hZTL1 and ZnT5 (Kambe et al. 2002) reveals that they are splice variants of the same gene and that the assay used previously to examine gene regulation is non-specific for the different transcripts. ZnT5 has additional exons upstream of the first exon of hZTL1, therefore we hypothesise that the two transcripts are expressed from alternative, tissue-specific promoters of which only the promoter active in intestine is zinc responsive. Consistent with this hypothesis, we present evidence that the genomic region immediately upstream of the first exon of the hZTL1 transcript is inactive as a promoter in JAR cells and that an exon unique to the ZnT5 transcript is expressed in JAR but not Caco-2 cells.
A 2899 bp region of the putative hZTL1 promoter region, including the 5Ô-most end of the cDNA, was subcloned into pBlue TOPO upstream of the E. coli β-galacotosidase reporter gene to give the plasmid pC2899. The plasmid pC2899 was transfected into JAR cells for transient expression. The plasmid pcDNA3.1/lacZ, including the E. coli β-galactosidase gene expressed from the strong CMV promoter, was used as a positive control and pBlue TOPO without insert was included as a negative control. β-Galactoside activity was measured in cell lysates prepared 48 h post-transfection. Data are means ± S.E.M. in arbitrary units; n = 6; statistical analysis was by one-way ANOVA followed by Bonferroni’s multiple comparisons test. β-Galactosidase activity expressed from pC2899 was not different from that of the negative control (1.58 ± 0.02 compared with 3.04 ± 0.34); however, activity of the positive control (39.0 ± 3.74) was significantly greater than both the negative control and pC2899 (P < 0.001). Addition of 100 µM ZnCl2 24 h post-transfection did not induce promoter activity. Analysis of RNA from Caco-2 and JAR cells by RT-PCR followed by sequencing, using primers specific for a region unique to the ZnT5 transcript, revealed expression only in JAR cells.
These data are consistent with expression of ZnT5 in JAR cells from a promoter region other than the region of genomic DNA immediately 5Ô of the first exon of hZTL1. We suggest that Caco-2 cells express hZTL1 and that the promoter for hZTL1 expression is distinct from the ZnT5 promoter and, unlike the ZnT5 promoter, is transcriptionally activated by zinc.
This work was funded by BBSRC grant 13/D11912 and by a BBSRC studentship.