Slc7a11 gene controls production of pheomelanin pigment and proliferation of cultured cells

Abstract

In mammals, >100 genes regulate pigmentation by means of a wide variety of developmental, cellular, and enzymatic mechanisms. Nevertheless, genes that directly regulate pheomelanin production have not been described. Here, we demonstrate that the subtle gray (sut) mouse pigmentation mutant arose by means of a mutation in the Slc7a11 gene, encoding the plasma membrane cystine/glutamate exchanger xCT [Kanai, Y. & Endou, H. (2001) Curr. Drug Metab. 2, 339-354]. A resulting low rate of extracellular cystine transport into sut melanocytes reduces pheomelanin production. We show that Slc7a11 is a major genetic regulator of pheomelanin pigment in hair and melanocytes, with minimal or no effects on eumelanin. Furthermore, transport of cystine by xCT is critical for normal proliferation, glutathione production, and protection from oxidative stress in cultured cells. Thus, we have found that the Slc7a11 gene controls the production of pheomelanin pigment directly. Cells from sut mice provide a model for oxidative stress-related diseases and their therapies.

Fig. 1.

Positional cloning of the sut gene. (A) High-resolution genetic map of the sut genetic interval. (B) High-resolution physical map. Five known genes in the sut interval are listed with arrowheads indicating transcriptional orientations. (C) Region of the genome between Pcdh18 and Slc7a11. The sut deletion is depicted by a dashed line. The sizes of introns (lines), exons (boxes), and intergenic regions (bold lines) are given in base pairs. (D) Transcripts of Slc7a11 in sut and control (C3H/HeSnJ) brains identified by 3′ RACE. In sut, Slc7a11 utilizes an alternative exon 12 (E12′, open box) from beyond the deleted region. (E) Predicted proteins, xCT-sut, and xCT-C3H, encoded by transcripts in sut and C3H/HeSnJ respectively. The underlined bold residues in xCT-C3H are conserved across species.

Fig. 2.

Altered regulation of Slc7a11 in sut mutants. (A) Northern blots of poly(A)-mRNA were probed with labeled Slc7a11 (Upper) and β-actin (Lower) cDNAs. (B) One transgenic rescued sut/sut agouti pup (positive for BAC RP23-22O3, which contains Slc7a11) plus a sut/sut littermate and a heterozygous (sut/+) control. Note agouti color in BAC-positive pup compared with the gray color of sut/sut pup. (C) There is a near-complete loss of yellow/red (pheomelanin) pigment in sut/sut, A^y/a mutants compared with the +/+, A^y/a controls.

Fig. 3.

Cystine transport and growth are reduced in cultured sut cells. (A Upper)[³⁵S]Cystine transport was measured in melanocytes and macrophages from +/+ (▴) and sut/sut (⋄) mice cultured under normal oxidizing conditions. Effects of 2.5 mM glutamic acid [melan-a +/+ melanocytes (•); sut-melanocytes (□)] and 2.5 mM arginine [melan-a +/+ melanocytes (♦); sut-melanocytes (○)] on the transport of [³⁵S]cystine were assayed at 5 min. (A Lower)[³H]Leucine transport into melanocytes and macrophages. Values are the average of duplicates from a representative experiment. (B) Growth of sut cells is attenuated under oxidizing culture conditions. Fibroblasts and melanocytes were grown with (+) and without (-) βME. Values are the average of duplicates from a representative experiment.

Fig. 4.

Loss of Slc7a11 expression causes loss of glutathione. Total glutathione was measured in five separate analyses of melan-a and sut melanocytes grown in the absence (-βME) or presence (+βME) of βME (See Table 2 legend for details). Total glutathione was >93% the reduced GSH form in all analyses. Values are mean ± SEM. ^***, P ≤ 0.001. ND, not detected.

Fig. 5.

Microscopy of wild-type (melan-a, A and C) and sut-mutant (B and D) melanocytes. (A and B) Bright-field images. (C and D) Dopa-stained cells; arrows in D indicate the perinuclear distribution of dopa reaction product in sut cells.

Fig. 6.

Ultrastructure of l-dopa-stained sut melanocytes. Melanocytes from sut mice were cultured in the absence (A) and presence (B) of βME, stained with dopa, and observed in the electron microscope. Arrows in A indicate increased staining of tubular/vesicle structures, possibly the trans-Golgi network, typically observed in sut cells cultured without βME.