We have recently cloned a functionally active zinc transporter, hZTL1, that is expressed at the apical membrane of the human intestinal Caco-2 cell line. We report here on the effect of changes in zinc concentration on levels of hZTL1 mRNA in Caco-2 cells and the human placental cell line JAR.

Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % (v/v) fetal calf serum, 60 µg ml^-1 gentamicin, 2 mM L-glutamine and 1 % (v/v) non-essential amino acids over four passages. The Zn²⁺ concentration of the nutrient medium was progressively increased from 20 to 100 µM by adding additional ZnCl₂ to confluent monolayers, thus avoiding toxicity. Total RNA was harvested after maintenance for 7 days at 100 µM ZnCl₂. JAR cells were cultured in RPMI-1640 supplemented with 10 % (v/v) fetal calf serum, penicillin/streptomycin (100 units ml^-1 and 100 µg ml^-1, respectively) and 2 mM L-glutamine. The zinc concentration of the nutrient medium was increased from 3 to 100 µM by the addition of ZnCl₂ to cells 1 day post-seeding. Total RNA was harvested after maintenance for 6 days at 100 µM ZnCl₂. Levels of hZTL1 mRNA in zinc supplemented cells and co-passaged controls were compared by semi-quantitative RT-PCR.

All data are expressed as means ± S.E.M. in arbitrary units as a ratio of 18s rRNA levels; n = 6; statistical analysis by Student’s unpaired t test. Increasing the ZnCl₂ concentration of the nutrient medium from 20 to 100 µM in Caco-2 and JAR cells had no effect on hZTL1 mRNA levels (0.20 ± 0.03 compared with 0.24 ± 0.02, P = 0.53 for Caco-2 cells, and 0.25 ± 0.03 compared with 0.25 ± 0.05, P = 0.93 for JAR cells at 20 and 100 µM ZnCl₂, respectively). For both cell lines, mRNA levels for metallothionein, well-established to be zinc induced (Andrews, 2000), were increased at 100 µM ZnCl₂ compared with 20 µM ZnCl₂ (1.07 ± 0.04 increased to 1.86 ± 0.08 for Caco-2 cells and 0.77 ± 0.05 increased to 2.58 ± 0.12 for JAR cells; P < 0.0001).

There are two copies of the hZTL1 gene in the human genome, on chromosomes 5 and 17. Whilst the exon structure of both copies is identical and regions 5Ì of the open reading frame are conserved, there are differences in the intron regions. The promoter region includes a single core consensus sequence for the zinc-activated transcription factor MTF-1 (Andrews, 2000). The data presented here indicate that hZTL1 is not regulated by MTF-1 but, rather, is expressed at constitutive levels over a wide range of supraphysiological zinc concentrations.

This work was funded by BBSRC grant 13/D11912 (D.F. and J.C.M.) and by a BBSRC studentship (R.M.R.).