Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells.

DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller V(max) for iron uptake compared with control cell lines. One clone was further characterized and found to display an approximately 50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism.

[1]  Y. Berger,et al.  The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications , 1995, Cell Biology and Toxicology.

[2]  J. Bressler,et al.  Involvement of DMT1 in uptake of Cd in MDCK cells: role of protein kinase C. , 2001, American journal of physiology. Cell physiology.

[3]  F. Elisma,et al.  Evidence for cadmium uptake through Nramp2: metal speciation studies with Caco-2 cells. , 2001, Biochemical and biophysical research communications.

[4]  H. Dietz,et al.  Inhibition of fibrillin 1 expression using U1 snRNA as a vehicle for the presentation of antisense targeting sequence. , 1997, Human molecular genetics.

[5]  H. Dietz,et al.  Reversion of human glioblastoma malignancy by U1 small nuclear RNA/ribozyme targeting of scatter factor/hepatocyte growth factor and c-met expression. , 1999, Journal of the National Cancer Institute.

[6]  P. Aisen,et al.  Chemistry and biology of eukaryotic iron metabolism. , 2001, The international journal of biochemistry & cell biology.

[7]  H. Hauser,et al.  Characterization of an integral protein of the brush border membrane mediating the transport of divalent metal ions. , 2000, Biophysical journal.

[8]  L. Goldman,et al.  Iron deficiency associated with higher blood lead in children living in contaminated environments. , 2001, Environmental health perspectives.

[9]  W. Stremmel,et al.  Iron uptake by human upper small intestine microvillous membrane vesicles. Indication for a facilitated transport mechanism mediated by a membrane iron-binding protein. , 1990, The Journal of clinical investigation.

[10]  E. Gunter,et al.  No difference in iron status between children with low and moderate lead exposure. , 1999, The Journal of pediatrics.

[11]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[12]  R. Leggett,et al.  Blood lead concentrations in hereditary hemochromatosis. , 1994, The Journal of laboratory and clinical medicine.

[13]  M. Portnoy,et al.  Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells. , 2002, Biochemical and biophysical research communications.

[14]  R J Simpson,et al.  An Iron-Regulated Ferric Reductase Associated with the Absorption of Dietary Iron , 2001, Science.

[15]  D. Bannon,et al.  Graphite furnace atomic absorption spectroscopic measurement of blood lead in matrix-matched standards. , 1994, Clinical chemistry.

[16]  A. Brownlie,et al.  Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter , 2000, Nature.

[17]  J. C. Smith,et al.  Reduction of Fe(III) is required for uptake of nonheme iron by Caco-2 cells. , 1995, The Journal of nutrition.

[18]  P. Gros,et al.  Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. , 1999, Blood.

[19]  M. Worthington,et al.  Functional properties of transfected human DMT1 iron transporter. , 2000, American journal of physiology. Gastrointestinal and liver physiology.

[20]  P. Campbell,et al.  Caco-2 Cell Line Used as an In Vitro Model to Study Cadmium Accumulation in Intestinal Epithelial Cells , 1997, The Journal of Membrane Biology.

[21]  S. Srai,et al.  Nramp2 Expression Is Associated with pH-dependent Iron Uptake across the Apical Membrane of Human Intestinal Caco-2 Cells* , 2000, The Journal of Biological Chemistry.

[22]  Stephan Nussberger,et al.  Cloning and characterization of a mammalian proton-coupled metal-ion transporter , 1997, Nature.

[23]  T. Simons.  Passive transport and binding of lead by human red blood cells. , 1986, The Journal of physiology.

[24]  N. Andrews,et al.  Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Vahter,et al.  Phlebotomy increases cadmium uptake in hemochromatosis. , 2000, Environmental health perspectives.

[26]  A. Zweibaum,et al.  A and H blood group antigens as markers of sucrase-isomaltase from the enterocyte-like differentiated human colon carcinoma cell lines HT-29 and Caco-2. , 1987, Cancer research.

[27]  B. Elsenhans,et al.  Iron retention and distribution in the cadmium-induced iron deficiency. , 1985, Ecotoxicology and environmental safety.

[28]  M. Zuker.  Computer prediction of RNA structure. , 1989, Methods in enzymology.

[29]  W. Gerlach,et al.  Simple RNA enzymes with new and highly specific endoribonuclease activities , 1988, Nature.

[30]  M. Yoshioka,et al.  Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2. , 1990, Gastroenterology.

[31]  H. Saito,et al.  Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2. , 1993, The American journal of physiology.

[32]  B. Lönnerdal,et al.  DMT1 gene expression and cadmium absorption in human absorptive enterocytes. , 2001, Toxicology letters.