Regulation of tyrosinase in human melanocytes grown in culture.

Abstract

Tyrosinase, the enzyme that controls the synthesis of melanin, is a unique product of melanocytes. Normal and malignant human melanocytes grown in culture were used to study the factors that regulate the expression of tyrosinase. Immunoprecipitation experiments showed that newly synthesized tyrosinase appeared as a protein with an apparent molecular weight of 70,000 that was processed to a protein with an apparent molecular weight of 80,000. Neither tunicamycin nor 2-deoxy-D-glucose inhibited this conversion, suggesting that Oglycosylation is the major biochemical event in the posttranslational modification of tyrosinase. Agents that stimulated the proliferation of normal melanocytes also stimulated tyrosinase activity. Melanocytes with low levels of tyrosinase activity synthesized less tyrosinase, processed the enzyme more slowly, and degraded it more rapidly than melanocytes with high levels of tyrosinase activity. We conclude that tyrosinase activity in cultures of human melanocytes derived from different donors is determined predominantly by its abundance.Epidermal melanin is synthesized by melanocytes via oxidation of tyrosine. The amount of melanin in the epidermis is determined, at least in part, by the activity of tyrosinase. For example, the activity of tyrosinase is higher in foreskins taken from black babies than in foreskins taken from caucasian babies (31); and melanocytes in patients with some forms of albinism have extremely low or no detectable tyrosinase activity (17). Knowledge of factors that regulate tyrosinase is important because melanin protects the skin from harmful solar radiation. In murine melanoma cells grown in vitro the activity of tyrosinase is at least partially regulated by the intracellular levels of cyclic adenosine monophosphate (cAMP). Exposure of these cells to substances that increase the intracellular levels of cAMP also causes an increase in tyrosinase activity and melanin content (1, 6-9, 22, 33, 38).Recently, a method for propagating normal human melanocytes in vitro (5) was developed. This achievement made it possible to study the control of tyrosinase activity in nonmalignant human melanocytes and to compare their responses with those of murine and human melanoma cells. Our experiments show that the activity of tyrosinase in cultured melanocytes is stimulated by agents that stimulate the growth of the cells and is determined by the rate of synthesis, processing, and degradation of the enzyme.

Tissue distribution and differential expression of melanocortin 1 receptor, a malignant melanoma marker

“…There is accumulating evidence that a-MSH, which is the ligand for MC1R, besides being a hormone involved in pigmentation (Halaban et al, 1983;Kameyama et al, 1989) also plays a crucial role in the regulation of immune and inflammatory reactions (Blalock, 1999;Luger et al, 2000). One consequence of this could be that MC1R also could be expressed on haematopoietic cells.…”

Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells

“…Two of the melanoma cell strains persistently were amelanotic (YUSIT1 and YUSAC2), and two, for yet unknown reasons, fluctuated between being amelanotic and slightly melanotic (501 mel and YUGEN8). These four strains represented phenotypes seen in at least 10 other melanoma cell lines (1,3,23,24). Despite having low levels of tyrosinase protein, they maintained tyrosinase mRNA levels similar to those of normal melanocytes (unpublished results) and expressed other melanogenic proteins essential for the formation of melanin (data not shown).…”

“…Pulse-chase experiments were performed and analyzed with slight modifications as described (1). In brief, cells were pulse-labeled (15 min) with [ 35 S]Protein Labeling Mix (EXPRE 35 S 35 S, 0.8 mCi͞ml; DuPont͞NEN) in methionine͞ cysteine-free medium and either collected immediately or after a chase incubation in nonradioactive medium for the indicated time.…”

“…Tyrosinase assays were performed as described (1). One unit of Immunoprecipitation reaction mixtures contained 3.6 ϫ 10 7 cpm in trichloroacetic acid-precipitable material (lanes 1-11) or 2 ϫ 10 7 cpm (lanes 12-15).…”

Human melanocyte senescence and melanoma susceptibility genes

“…Stimulation of pigment cell differentiation by cAMP agonists is a long-studied topic (e.g. Wong and Pawelek, 1975;Halaban et al, 1983;Bennett, 1989;Haddad et al, 1999;Busca`and Ballotti, 2000). Pathways include both stimulation of transcription of specialized melanocytic proteins (for reviews, see Busca`and Ballotti, 2000;Goding, 2000) and post-translational activation of the key melanosomal enzyme tyrosinase (Wong and Pawelek, 1975).…”