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Molecular and Cellular Biology, April 2004, p. 3396-3403, Vol. 24, No. 8
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.8.3396-3403.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Fabien Murisier,1, Anita Rossier,1, Alexandre Moulin,2 and Friedrich Beermann1*
Molecular Oncology, Swiss Institute for Experimental Cancer Research, National Center of Competence in Research, 1066 Epalinges,1 Institute of Pathology, University of Lausanne, 1005 Lausanne, Switzerland2
Received 27 October 2003/ Returned for modification 7 December 2003/ Accepted 31 January 2004
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ABSTRACT |
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INTRODUCTION |
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The synthesis of the melanin pigment is ascertained by tyrosinase (EC 1.14.18.1), the key enzyme in pigment synthesis, and two related enzymes, tyrosinase-related protein 1 (TYRP1) and dopachrome tautomerase (DCT) (EC 5.3.2.3), which are strongly implicated in eumelanin synthesis (10). The switch between the eumelanin and the pheomelanin pathways is thought to depend on the presence of cysteine. Thus, in the absence of cysteine, dopaquinone, the product of tyrosinase action, is transformed to cyclodopa (leucodopachrome) and then to dopachrome (and DOPA). Dopachrome gives rise to 5,6-dihydroxyindole (DHI) or is enzymatically metabolized by DCT, yielding 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (40). These dihydroxyindoles might then be oxidized to eumelanin. Since DHI and its oxidation products are toxic to cells (33, 42), it was thought that DCT is required to prevent a too-high level of toxic intermediate from being produced within the cell.
The gene coding for DCT (Dct) has been identified in many species, including nonmammalian vertebrates such as chickens, Xenopus, and puffer fish (1, 6, 24). In mice, in situ hybridization assays and transgenic lacZ reporter gene assays have provided evidence that Dct is specifically expressed in cells of the pigment cell lineage, namely melanocytes and melanoblasts, and cells of the RPE (25, 36, 43). Onset of expression is at about embryonic development day 9 (E9) in RPE and at E11 in migrating melanoblasts and thus is somewhat earlier than that of the two related genes of the family, tyrosinase and Tyrp1. Moreover, in adult mouse skin, only Dct seems to be expressed in melanocyte stem cells residing in the bulge of the hair follicle, whereas Tyrp1 and the tyrosinase gene are restricted in expression to more differentiated cells and transiently amplifying cells (3, 28). Apart from the rather expected expression within cells of the pigment cell lineage, Dct is expressed in the developing telencephalon (25, 36, 43) and it was speculated that it might serve as detoxifying enzyme within neuronal tissue.
Mutations at all three tyrosinase gene family members, Tyr (tyrosinase), Tyrp1 (TYRP1), and Dct (DCT) have been found in mice (http://www.informatics.jackson.org). At the Tyr locus, the mutations range from absence of any pigmentation in the albino allele (Tyrc) to phenotypes with only slightly reduced pigmentation, such as, for example, chinchilla (Tyrc-ch). Similarly, mutations at Tyrp1 affect coat color, with the brown (Tyrp1b) mutation at the extreme representing no TYRP1 activity left. It is important to note that some of these mutations (Tyrp1b and Tyrc) still enable protein production and the phenotype is rather due to intracellular mislocalization and retention of the proteins in the endoplasmic reticulum (19, 39). Nevertheless, null mutations of both Tyrp1 and Tyr have been created by large deletions in the course of mutagenesis screens, and the corresponding phenotypes are identical to the Tyrc or Tyrp1b mutation, respectively. In contrast, only three mutations (Dctslt, Dctslt-J, and DctSlt-Lt) are known at the Dct locus, all of which lead to pigmentation phenotypes (4). They are all point mutations and still enable expression of mutant protein. It has even been argued that Dctslt, which depicts the best-characterized phenotype, might retain some DCT activity (4, 22). It therefore remained an open question of whether the absence of DCT activity leads to a coat color phenotype or is of importance in other Dct-expressing tissues, where it might contribute essential detoxifying function. To address this issue, we genetically inactivated the Dct gene by gene targeting in embryonic stem (ES) cells.
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MATERIALS AND METHODS |
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clone of 129/SV DNA had been cloned into pBluescript and was provided by Peter Budd and Ian Jackson (4). This fragment contained 3.2 kb of 5' sequence and exons 1 to 4 of the mouse Dct gene. A 4.5-kb EcoRI-XhoI fragment of the first intron was used for 3' homology, and, following several intermediate cloning steps to remove or add restriction sites, finally cloned as an EcoRI-BamHI fragment into the EcoRI and BamHI sites of the targeting vector, pPNT (41). A 1.1-kb BglII-StuI fragment of the Dct promoter was isolated from a Dct promoter plasmid (NotI-SacI fragment) (4), and, following different intermediate cloning steps, finally linked to a 1.6-kb MluI-SalI fragment of Cre recombinase (from plasmid pMC-Cre) (17). The resulting 1.1-kb Dct-Cre expression plasmid was cloned as a NotI-XhoI fragment in the corresponding sites of pPNT. GS-1 ES cells (129S6/SvEv) were electroporated with the NotI-linearized targeting vector and selected for resistance to G418 and ganciclovir (21). Southern blot analysis was performed following PstI digestion of genomic ES cell DNA using a 32P-labeled probe (1.5-kb HindIII-BglII fragment of the Dct gene promoter). Out of 166 double-resistant clones, 4 were correctly targeted. We injected two independent clones into blastocysts from C57BL/6 mice and crossed the resulting chimeras back to C57BL/6 mice to obtain heterozygous Dct mutant mice, Dcttm1(Cre)Bee. Expression of Cre recombinase from the Dcttm1(Cre)Bee mutant mice has been described elsewhere (18) and is not discussed further in this article.
Genotyping was performed using DNA isolated from tail biopsies and analyzed by PCR using three primers (Dct sense, 5'-TTGAGAGGAGAGGAAAGGGC-3'; Dct antisense, 5'-CACGCCATCCAAGGTCATGC-3'; and Cre antisense, 5'-CATTGCTGTCACTTGGTCGT-3'), resulting in a Dct knockout-specific PCR product (
600 bp) and a product specific for the endogenous gene (289 bp). To control for the absence of exon 1 coding sequence in knockout mice, a separate set of primers was used (Dct sense, 5'-CTGCGGAATTCTGCTCA-3'; and Dct antisense, 5'-TTCTCGGCCATTGCTCA-3'), resulting in a 224-bp fragment. For reverse transcription-PCR, total RNA was isolated from 2-day-old mouse skin. RNA was treated with DNase, and the first cDNA strand was synthesized with Superscript (Life Technologies). PCR was performed using primers situated in exons 4 and 5 of Dct (exon 4, Dct sense, 5'-ATGAGTCCTTTGCGTTGCCC-3'; and exon 5, Dct antisense, 5'-TGAAGCTGAAGGTAGAGT-3'), amplifying a 333-bp fragment. The integrity of the cDNA was controlled by amplification using ß-catenin-specific primers (exon 2, ß-catenin sense, 5'-CGTGGACAATGGCTACTCAA-3'; and exon 4, ß-catenin antisense, 5'-CCCTCATCTAGCGTCTCAGG-3'), resulting in a 325-bp fragment.
Breeding experiments. Except for specific analyses, mice were kept nonagouti (a) and wild type at Tyrp1 (Tyrp1+) and Tyr (Tyr+) loci. Homozygous mutant mice on an agouti background (Aw) were obtained by interbreeding the first chimeric offspring. Homozygous mutant mice with a mutation at the Tyrp1 locus (Tyrp1b) and/or the Tyr locus (Tyrc) were obtained by breeding Dcttm1(Cre)Bee homozygous mutant mice (a/a, Tyr+/Tyr+, and Tyrp1+/Tyrp1+) with BALB/c mice (A/A, Tyrp1b/Tyrp1b, and Tyrc/Tyrc). The resulting offspring were intercrossed to generate mice which are brown (Tyrp1b/Tyrp1b) and nonagouti (a) or agouti (A) but still carry the Dcttm1(Cre)Bee allele. Mice [Dcttm1(Cre)Bee/+, Tyrp1b/Tyrp1b, Tyr+/Tyr+, or Tyr+/Tyrc] were bred with each other to obtain both brown mutant mice deficient in Dct [Dcttm1(Cre)Bee/Dcttm1(Cre)Bee, Tyrp1b/Tyrp1b] and brown albino mice deficient in Dct [Dcttm1(Cre)Bee/Dcttm1(Cre)Bee, Tyrp1b/Tyrp1b, and Tyrc/Tyrc]. For comparison with slaty (Dctslt), mice with homozygous mutation at this allele (Dctslt; kindly provided by L. Lamoreux, Austin, Tex.) and kept on a C57BL/6 background were bred to Dcttm1(Cre)Bee homozygous mutant mice (a/a, Tyr+/Tyr+, and Tyrp1+/Tyrp1+). The resulting offspring were mated to each other to generate littermates of the three genotypes Dctslt/Dctslt, Dcttm1(Cre)Bee/Dcttm1(Cre)Bee, and Dctslt/Dcttm1(Cre)Bee.
Primary melanocyte culture. To isolate primary melanocytes (14), a piece of shaved skin (adult skin, 10 to 15 cm2; 1-week-old, whole-back skin) was washed in phosphate-buffered saline (PBS), cleaned of fat, and incubated overnight at 4°C in 0.25% trypsin. Dermis was separated from epidermis, minced with scissors, and incubated for 3 to 5 h at 37°C in 10 ml of collagenase (0.1% [wt/vol]; Sigma). Following mixing (by pipetting up and down) and three rounds of centrifugation and resuspension in PBS (282 x g, 5 min), the pellet was resuspended in primary melanocyte medium (5% fetal calf serum in RPMI 1640 with Glutamax [Gibco], containing 23-ng/ml 3-isobutyl-1-methylxanthine [Sigma], 10-µg/ml bovine pituitary extract [Sigma], and 10-µg/ml insulin [Sigma]) and placed on 60- to 100-mm-diameter cell culture dishes. G418 (50 µg/ml) and 35-ng/ml tetradecanoylphorbol-13-acetate (TPA [Sigma]) were added after 24 h. Initially, we encountered problems in obtaining melanocytes from Dct knockout mice, which might be due to melanin production and accumulation of toxic intermediates such as DHI: therefore, we added catalase (100 U/ml) to the medium (42). In later experiments, catalase was no longer added to the culture medium. Proliferation was measured on 3-week-old cultures derived from 1-week-old mice. A total of 6 x 105 to 10 x 105 cells were plated on a 10-cm dish, and the total cell number per dish was determined after 8 days, following trypsinization.
All antibody stainings were performed at room temperature in 24-well plates. Cells were grown on coverslips coated with poly-D-lysine (0.1 mg/ml) (Sigma) and fixed for 15 min in 2% paraformaldehyde. After being washed with PBS, the cells were permeabilized with Triton X-100 (0.1% [wt/vol]) and 1% bovine serum albumin (BSA) for 10 min at room temperature. Nonspecific binding sites were blocked for 30 min with 5% BSA and 5% fetal calf serum in PBS. The cells were then incubated for 90 min at 37°C with the primary antibody (anti-PEP7, anti-PEP8, and anti-PEP1) and for 30 min with the secondary antibody (CY3-coupled donkey anti-rabbit immunoglobulin G). Nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole) for 10 to 20 min at room temperature. Coverslips were mounted on slides with 1,4-diazobicyclo(2,2,2)-octane (DABCO). Confocal microscopy of cells grown on coverslips was performed according to standard procedures (12).
Total melanin. Melanin was extracted from dorsal hair (1 to 4 mg) of 4- to 8-week-old mice by treatment with alkali (1 ml of 1 M NaOH, 4 h at 85°C). Relative net melanin content (per gram of hair) was determined by spectrophotometric determination of A475. Each hair sample was measured in triplicate. Data are presented as means ± standard error. Statistical significance was assessed by the t test.
Western blot. Total protein extracts were isolated from adult mouse skin. Western blots were performed as described previously (11, 12). Briefly, 100 to 150 µg of protein was boiled for 5 min in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and electrophoresed on sodium dodecyl sulfate-8% polyacrylamide gels. The proteins were transferred to a nitrocellulose membrane in a mixture of 20% methanol, 40 mM glycine, and 50 mM Tris base. After transfer, membranes were saturated overnight with PBS containing 4% milk, 1% BSA, and 0.25% Tween 20. Membranes were incubated with the primary antibody (anti-PEP1 or anti-PEP8) overnight at 4°C, washed, and incubated with the secondary antibody (horseradish peroxidase, mouse anti-rabbit) for 30 min. Finally, the membrane was washed again, and immunocomplexes were visualized by a chemiluminescent reaction (ECL; Amersham Life Science, Otelfingen, Switzerland).
Histology and immunohistochemistry. For histological analysis, organs of adult mice were dissected, fixed overnight in 4% paraformaldehyde, and embedded in paraffin. Seven-micrometer sections were stained with hematoxylin and eosin. For immunofluorescence, cryostat sections of E14.5 embryos were fixed for 3 min in ice-cold acetone, blocked in PBS-1% BSA, and incubated for 1 h at room temperature with primary antibodies. DCT and tyrosinase were detected with anti-PEP8 and anti-PEP7 antibodies (kindly provided by V. Hearing). The sections were incubated for 30 min at room temperature with the secondary antibody (donkey anti-rabbit CY3), washed three times in PBS, and mounted in Citifluor (type 40-88; Aldrich-Chemie, Steinheim, Germany).
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RESULTS |
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Culture of Dct-deficient primary melanocytes. Even though we have not seen any evidence for changes in melanocytes in the living mouse, toxic effects might be tampered with or compensated for by surrounding keratinocytes or fibroblasts. We hence isolated primary melanocytes from skin of Dct knockout mice. In all of these experiments, melanocyte numbers were not obviously different when beginning with the isolation procedure, at a point at which fibroblasts and keratinocytes are still present. Dct-deficient primary melanocytes could be isolated and seemed not to be affected in viability or proliferation (Fig. 4a and i). When counting cell numbers in such primary cultures, viability of Dct-deficient melanocytes was not reduced compared to that in wild-type melanocytes (not shown). Equally, primary melanocytes were grown from albino mice lacking tyrosinase and TYRP1 function (Tyrc/Tyrc, Tyrp1b/Tyrp1b) and with either homozygous or heterozygous mutation of the Dct knockout allele. Again, deficiency of Dct did not affect primary melanocyte cultures (Fig. 4b and c). Cultures of pigmented Dct knockout melanocytes were then used for immunofluorescence analysis, which confirmed the absence of DCT expression (Fig. 4f) and demonstrated the normal expression and localization of tyrosinase and TYRP1 (Fig. 4d and e), depicting the punctate pattern, with perinuclear density representing melanosomes and trans-Golgi network (TGN) localization (Fig. 4g and h). The results show that Dct-deficient melanocytes grow in culture and suggest that absence of DCT does not affect localization of tyrosinase and TYRP1.
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DISCUSSION |
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DCT has been shown to be an enzyme of the eumelanin biosynthetic pathway (22, 40). In presence of the enzyme, dopachrome is metabolized to DHICA. The part of dopachrome which is not metabolized will be converted spontaneously to DHI. Most of these intermediate substances, such as DHI, DHICA, and, equally, DOPA, are cytotoxic in cell culture (32, 33, 42), which is mainly due to oxidation products, such as H2O2. For example, antiproliferative effects of DHI could be abrogated in the presence of catalase (42). More DHI should be produced in primary melanocytes lacking DCT, thus causing cytotoxic effects. In consequence, when we began with isolation of primary melanocytes from pigmented Dct-deficient mice, we sometimes encountered problems in proliferation and growth (not shown) like those described for exogenously added DHI (42). Since similar problems have been mentioned for melanocytes of Dctslt mice (9), we argued that this effect is due to the accumulation of toxic by-products, including H2O2, and added catalase to the melanocyte culture medium, which helped to improve viability. This effect was clearly linked to pigment production and to the absence of DCT, since albino melanocytes lacking DCT were not affected and always grew as controls. In later experiments, we started isolation with more cells and avoided inclusion of cellular debris and hairs. Under such conditions, Dct knockout melanocytes grew and proliferated similarly with or without catalase in the medium (Fig. 4). In vivo, in Dct knockout mice, we have not seen any obvious decrease in melanocyte numbers. When isolating primary melanocytes from mice, no difference in number was observed. It is feasible that, in skin, interaction with keratinocytes and fibroblasts might somehow prevent accumulation of cytotoxic products.
Besides pigment cells, Dct expression is found within the developing telencephalon (31, 36), and it had been suggested that DCT might exert detoxifying functions, similar to the potency to avoid reactive oxygen species generated by autooxidation of melanin precursors (4). Brain sections of adult Dct knockout mice revealed no difference from wild-type mice and thus make it unlikely that absence of DCT has any effect in the developing telencephalon. A toxic effect would have been apparent in our experiment, even though we cannot completely exclude having missed minor effects or that brains of knockout mice are more sensitive to toxic insult. It might nevertheless be feasible that the expression of Dct in telencephalon is without functional relevance.
Interestingly, the 5' regulatory region of only 6 kb of the mouse tyrosinase gene promoter leads to expression of the reporter gene lacZ in the developing telencephalon (38), without obvious expression of mRNA or detectable enzyme activity (15, 20). Use of an enlarged 5' regulatory region, encompassing the tyrosinase enhancer/locus control region, apparently prevents this expression pattern (5, 13, 34). It might thus be possible that tyrosinase expression in forebrain has to be inhibited, and, hence, additional control elements have evolved. On the other side, expression of Dct in forebrain might be of no functional relevance and does not require to be suppressed.
The Dct locus was formerly called the slaty locus, according to the sole mutation known at this locus (16). In the meantime, two other mutations have been identified, DctSlt-lt (slaty light), with a coat color effect already evident in heterozygotes, and Dctslt-2J, which is similar to the original slaty (Dctslt) mutation (4). This mutation, which changes an arginine to a glutamine in the first copper binding domain, was reported to yield about 10 to 30% of wild-type DCT activity in eye extracts (22). In contrast, when a Dct cDNA carrying the slaty mutation was expressed in cells in culture, no enzymatic activity was detected. According to Western blots, protein was produced from both the wild-type and the slaty mutant (Dctslt) cDNA, even though the antibody reacted less with the protein encoded by the mutant cDNA (23). From our data, we suggest that the slaty mutation leads to a more severe reduction in melanin than the Dct knockout. All three members of the tyrosinase family are type 1 membrane-bound melanosomal glycoproteins with similar structural features, and all three are believed to interact with one another in melanosomes (30). TYRP1 and DCT might act together to modulate tyrosinase activity (26). Thus, a nonfunctional but processed DCT protein might prevent tyrosinase from being fully active and/or reaching the melanosome. Along this line, it is now clear that mutations in tyrosinase and TYRP1 affect the association of each with calnexin and Bip, thus leading to retention of mutant products in the endoplasmic reticulum (39). Even though the effect seems to be weaker, association of DCT with calnexin was slightly increased in a tyrosinase or TYRP1 mutant situation. From these experiments, it was concluded that tyrosinase and TYRP1, as well as, to some extent, DCT, associate. A mutation in any of these genes might thus affect maturation and degradation of the others (39). DCT was reported to have a maturation pathway distinct from tyrosinase and TYRP1 and is not only confined to melanosomes but distributed perinuclearly in the TGN (27). Hence, DCT might have additional regulatory function within the melanocyte (27). Consequently, the point mutation in the copper binding domain of the slaty (Dctslt) mutation might not only affect DCT activity within the melanosome but also might affect interaction with yet unknown proteins within the TGN.
DCT is expressed in most melanoma cells and is used as melanoma differentiation antigen (37). It was even reported that the presence of DCT in melanoma is correlated with resistance to certain chemotherapeutic agents (8). Thus, it is possible that the absence of DCT [Dcttm1(Cre)Bee] or altered DCT protein conformation and/or localization (Dctslt or DctSlt-Lt) will affect melanoma formation or melanoma sensitivity. This can now be tested by using Dct mutant and knockout mice following breeding with transgenic melanoma models.
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ACKNOWLEDGMENTS |
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The present work was supported by grant 3100-066796.01 (F.B.) from the Swiss National Science Foundation and by the National Center of Competence in Research Molecular Oncology, a research instrument of the Swiss National Science Foundation.
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FOOTNOTES |
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L.G., F.M., and A.R. contributed equally to this work.
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