Abstract. To survive and proliferate in pure culture, human melanocytes require basic fibroblast growth factor (bFGF) and cAMP. Without these factors, even in the presence of serum, the cells die. Melanocytes cultured in the presence of keratinocytes, however, survive for weeks without added bFGF and cAMP. We show here that the growth factor for melanocytes produced by human keratinocytes is bFGF because its activity can be abolished by neutralizing antibodies to bFGF and by a bFGF synthetic peptide that inhibits the binding of the growth factor to its receptor. The melanocyte mitogen in keratinocytes is cell associated and increases after irradiation with ultraviolet B. Northern blots reveal bFGF gene transcripts in keratinocytes but not melanocytes. These studies demonstrate that bFGF elaborated by keratinocytes in vitro sustains melanocyte growth and survival, and they suggest that keratinocyte-derived bFGF is the natural growth factor for normal human melanocytes in vivo.
The loss of tyrosinase, the key enzyme in melanin synthesis, has been implicated in the dedifferentiation of malignant melanocytes. The presence of tyrosinase transcripts and antigenic peptides in melanoma tumors prompted us to investigate whether the basis for the loss of the enzyme was proteolytic degradation. Toward this aim, we followed the kinetics of synthesis, degradation, processing, chaperone binding, inhibitor sensitivity, and subcellular localization of tyrosinase in normal and malignant melanocytes. We found that, in amelanotic melanoma cell lines, tyrosinase failed to reach the melanosome, the organelle for melanin synthesis, because it was retained in the endoplasmic reticulum (ER) and then degraded. Tyrosinase appeared mostly as a 70-kDa core-glycosylated, endoglycosidase H-sensitive, immature form bound to the ER chaperone calnexin and had a life-span of only 25% of normal. Maturation and transit from the ER to the Golgi compartment was facilitated by lowering the temperature of incubation to 31°C. Several proteasome inhibitors caused the accumulation of an Ϸ60-kDa tyrosinase doublet that was more prominent in malignant than in normal melanocytes and promoted, to various degrees, the maturation of tyrosinase in melanoma cells and the translocation of the enzyme to melanosomes. The appearance of ubiquitinated tyrosinase after treatment of normal melanocytes with N-acetyl-L-leucinyl-L-leucinal-L-norleucinal reinforced our notion that some tyrosinase is normally degraded by proteasomes. Proteolysis of tyrosinase by proteasomes is consistent with the production of antigenic tyrosinase peptides that are presented to the immune system by major histocompatibility complex class I molecules.Loss of pigmentation is observed in human melanomas in situ and in metastatic melanoma cells established in culture. The several studies designed to elucidate the basis for this phenotype have been focused on tyrosinase, the key enzyme of melanogenesis. In those in which both protein and mRNA levels were examined, a posttranscriptional regulation was implicated because, despite low or undetectable tyrosinase protein levels, tyrosinase mRNA was detected easily (refs. 1-4 and unpublished results). The latter was found in solid tumors, in cells in culture, and in blood-borne melanoma cells (5-7). The possibility that melanocyte-specific proteins were synthesized but later degraded was supported by the numerous reports identifying peptides derived from such melanogenic proteins as tyrosinase, TRP1͞gp75, and gp100͞Pmel 17 that serve as tumor antigens recognized by T cells of melanoma patients (see, for example, refs. 8 and 9 reviewed in ref. 10).Tyrosinase (70-80 kDa) is a type I membrane glycoprotein whose cDNA predicts a peptide of Ϸ58 kDa, a 28-amino acid cytosolic tail, and five putative N-glycosylation sites (refs. 2 and 11 and for review see refs. 12 and 13). Like other membrane glycoproteins, tyrosinase is processed in the endoplasmic reticulum (ER) by resident chaperones and enzymes (14,15). Reductions in me...
Multiple biopsy specimens from the skin of 28 patients with common vitiligo were examined by light and electron microscopy. The patients were grouped according to the activity of their disease: progressing, stable, repigmenting, and resistant to treatment with psoralen plus sunlight. Three biopsy sites were sampled from each patient: (W) a white spot; (I) the pigmented and white interface; and (P) normally pigmented skin 1-15 cm away from I. Control specimens were obtained from 17 persons without vitiligo. Two microscopic abnormalities were observed in the epidermis of the patients with vitiligo: deposits of extracellular granular material, and foci of vacuolar degeneration of basal and parabasal keratinocytes. The extracellular granular material appeared to be derived from the cytoplasm of vacuolated keratinocytes. The abnormalities were observed in greatest abundance in the normally pigmented skin of patients with rapidly progressing or stable disease. They were absent from repigmenting skin and from the skin of healthy controls. Epidermal infiltrates of mononuclear leukocytes were seen only in the normal pigmented skin of the 2 patients whose vitiligo was resistant to treatment. Our observations indicate that cellular degeneration and the generation of debris in vitiligo are not limited to melanocytes but include keratinocytes and probably whole epidermal melanin units. Our findings also indicate that the fine structure of the epidermis in normal-appearing skin is markedly altered by the disease process in patients with vitiligo.
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