Increased Expression of Dickkopf1 by Palmoplantar Fibroblasts Inhibits Melanocyte Growth and Differentiation

Yamaguchi Y, Itami S, Watabe H, Yasumoto K, Abdel-Malek ZA, Kubo T, Rouzaud F, Tanemura A, Yoshikawa K, and Hearing VJ. Mesenchymal-epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation. J Cell Biol 165: 275–85, 2004.

elanocytes, unique cells that specialize in producing the pigmented biopolymer melanin, give rise to visible color in the skin, hair, and eyes. These cells are derived from the neural crest during development (where they are termed melanoblasts): more than 120 genes have been identified that affect pigmentation during the development, migration, survival, and differentiation of these cells. Many of the genes have been cloned and associated with various inherited human pigmentary diseases. (See http://ifpcs.med.umn.edu/micemut.htm.) A number of genes (primarily encoding growth factors and transcription factors) are known to function during melanoblast development and specification, while others (primarily encoding enzymatic and structural components of the pigmentation pathway) are known to function during differentiation.

Figure 1. Melanocyte function in palm and trunk skin. A) Macroscopic view of hypopigmented palm and hyperpigmented arm skin; B) Fontana-Masson staining for melanin showing decreased pigment and increased thickness of palm skin; C) Increased expression of β-catenin (green) and melanocytes (stained red due to MART1, a melanosomal protein) in trunk skin; D) Expression of β-catenin in melanocytes co-cultured for 5 days with control (Ctrl) or with DKK1-transfected fibroblasts (left-most panel) and in melanocytes treated for 3 hours with or without 50 ng/mL DKK1 per hour (third panel from the left). β-actin is shown as a loading control. (Numbers indicate quantitation of bands.) E) Scheme illustrating the possible mechanism by which DKK1 decreases melanocyte growth and differentiation. LRP, Krn, Frizzled: Wnt receptors. MITF: a melanocyte transcription factor. DCT, MART1, TYR: melanosomal proteins.

It is obvious that skin on the palms of the hands and soles of the feet are relatively (often dramatically) less pigmented than the rest of the skin (Figure 1, part A), but to date, no genes have been identified that regulate such differences in pigmentation. This is an interesting and important topic because skin pigmentation is directly, but inversely, related to various types of skin cancer, and darkly pigmented skin is 15 times less susceptible to malignant melanoma as compared with lightly pigmented skin (and 50 times less susceptible to basal and squamous cell carcinomas). Many clinical terms are used to denote these types of skin, but in this summary, we will use the terms palm skin for lightly pigmented, palmoplantar skin and trunk skin for the darker, nonpalmoplantar skin.

Our laboratory has been involved in characterizing the regulation of skin pigmentation, resulting in the identification of many physiological and environmental factors (e.g., hormones and UV) that regulate melanocyte growth and differentiation; however, factors that regulate melanocyte density and function in the skin are only poorly understood. An important observation in this regard, made by our collaborators in Osaka, Japan, was that skin transplanted from trunk epidermis to deep wounds in the palm in the same individual gradually assumes the phenotype of the palm skin; that is, it becomes much thicker and much less pigmented, over the course of several months (Figure 1, part B). Indeed, melanocyte density in the trunk skin of Caucasians, Asians, and black/African Americans is virtually identical but is 5-fold lower in palm skin (Figure 1, part C). Since only the epidermis was transplanted, we hypothesized that the relevant regulatory factors were determined by the underlying dermis, which is made up primarily of fibroblasts. A number of recent studies by other groups have shown that different populations of fibroblasts can have widely differing but stable expression patterns of a large number of genes, so it seemed logical that fibroblasts in palm and in trunk skin might produce distinct factors that regulate melanocyte growth and function.

We established primary fibroblasts (derived from the palm and the trunk skin of individual donors) in culture and co-cultured them with melanocytes to recapitulate what was found surgically—that co-culturing with fibroblasts derived from palm skin markedly downregulates melanocyte growth and pigmentation. We used microarray and PCR analyses to examine their gene expression patterns. The majority of genes were comparable between the two populations, but a few genes that were differentially expressed were intriguing, notably, genes relating to two similar factors, dickkopf 1 (DKK1) and DKK3. Not only were they regulated inversely in the two fibroblast populations (from all 5 donors), but the mechanism of action of DKK1 is known to be through Wnt signaling. Wnt signaling has been known for some time to play an important role in regulating melanocyte growth and differentiation, particularly with respect to its effect on melanocyte transcription factors (such as MITF), which in turn regulate melanin production.

A series of molecular and biochemical approaches to stimulate or inhibit DKK1 function were used to observe the effects on melanocyte function. DKK1 (which is preferentially expressed by palm fibroblasts) remarkably inhibited melanocyte growth and also down-regulated pigment production. Inhibiting DKK1 function had the reverse effects, and this proved true whether the DKK1 originated from fibroblasts in co-culture (Figure 1, part D, left-most panel) or from recombinant protein added to the system (Figure 1, part D, third panel from the left), and the effects could be abrogated by the addition of a DKK1-specific inhibitory antibody (data not shown). As expected, we were able to show that the effects of DKK1 were in fact mediated via Wnt signaling, β-catenin expression, and MITF function. A summary of the DKK1–Wnt–β-catenin–MITF–TYR melanin cascade is shown (Figure 1, part E). Thus, our results provide a basis to explain why skin on the palms and the soles is generally hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in the palmoplantar area during human embryogenesis.

Yuji Yamaguchi, MD, PhD
Visiting Fellow
Osaka University Graduate
School of Medicine
Osaka, Japan

Vincent J. Hearing, PhD
Principal Investigator
Laboratory of Cell Biology
NCI-Bethesda, Bldg. 37/Rm. 2132
Tel: 301-496-1564
FAX: 301-402-8787
hearingv@nih.gov

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