Role and modulation of tyrosinase/tyrosinase related protein‐1 complex and PKC beta‐I in melanogenesis

In humans, pigmentation of the skin results from the synthesis and the distribution of melanin pigments. Epidermal melanocytes, residing at the basal layer of epidermis, are neural crest‐derived skin cells responsible for melanin pigment production [1]. Tyrosine and l ‐dihydroxyphenylalanine ( L ‐Dopa) serve as precursors for this complex biopolymer and darkened skin color is the result of increased and redistributed epidermal melanin [2]. The basal level of pigmentation is genetically predefined and modulated by many internal or external factors. One role of melanin is to protect the skin from harmful effects of sunlight and oxidative stress but unwanted pigmentation can produce a significant psychological stress [3]. Genetic and biochemical studies have identified several proteins that regulate melanin synthesis and the structural integrity of the melanosomal compartment. The best‐characterized melanogenic proteins are tyrosinase, a copper‐dependant glycoprotein, and its related proteins (tyrosine related protein‐1 and 2; TRP‐1 and TRP‐2) [4]. Human tyrosinase was cloned and sequenced [5] and the deduced amino acid sequences revealed that tyrosinase is composed of 511 amino acids with more than 90% of the enzyme inside the melanosome. Tyrosinase catalyzes the hydroxylation of tyrosine to Dopa, which is the rate limiting step of melanogenesis and the oxidation of Dopa to Dopaquinone. TRP‐1 has also been shown to play a crucial role in melanogenesis. The mature TRP‐1 is the most abundant melanosomal protein [6] and TRP‐1 shares with tyrosinase 70–80% nucleotide sequence homology and 40–45% amino acids identity. TRP‐1 is transmembrane protein spanning melanosomal membranes [7], involved in regulating melanosomal maturation [8] and in contrast with mice, TRP‐1 in human pigment cells do not display DHICA oxidase activity [9]. TRP‐1 was shown to influence tyrosinase activity by forming a complex stabilizing tyrosinase [10, 11] and recent study indicated ethnic variation in TRP‐1 expression suggesting its role in mediating differences in skin pigmentation in vivo [12]. In human cultured melanocytes, tyrosinase/TRP‐1 complex formation has been demonstrated and this complex formation was mediated by Protein Kinase C beta (PKC beta) [13]. Molecular cloning revealed that PKC is a multigene family of at least 12 isoforms. Among them, PKC beta has specifically been reported to activate tyrosinase and human melanogenesis [14] by phosphorylating serine residues in its cytoplasmic domain [15] indicating that this entity regulates human melanogenesis at an early stage. Loss of PKC beta prevents melanogenesis in cultured pigment cells [15] and topical application of pan‐PKC inhibitor has just been reported to reduce skin and hair pigmentation [16]. Among the principal manifestations of photodamage in Asian women's skin, solar lentigo is a common benign pigmented spot on sun‐exposed skin especially on the back of the hands, arms and on the forehead. For investigating this event, firstly, the evaluation on Japanese women's skin of the TRP‐1 expression in solar lentigo spots in comparison with normal skin was performed and secondly, the study of the messenger RNA expression inhibition coding for tyrosinase/TRP‐1 complex and/or PKC beta‐I on melanogenesis was carried out. This antisense strategy is based the use of sequence specific oligodeoxyribonucleotides targeted to messenger RNA for modulating the expression of the enzymes instead of a direct inhibition after enzyme synthesis. Antisense strategy is a very specific approach, occurring at an upstream level of melanogenesis, with activity at nanomolar concentration without cytotoxicity. This strategy, targeting messenger RNA, offers the only way to specifically inhibit PKC beta‐I, isoform expressed in melanocytes. Methods TRP‐1 expression study Biopsies (normal and solar lentigo area) were performed at Laboratoires Dermscan, (Lyon, France) in accordance with ethical committee procedure. The group consisted of 10 healthy Japanese women leaving in France with age ranging from 29 to 55 years. Biopsies were fixed in formaldehyde and embedded in wax. TRP‐1 detection was performed with Mel‐5 primary mouse monoclonal antibody (Signet, Dedham, U.S.A.) and a biotinylated antibody. Revelation was carried out with a standard phosphatase alkaline‐staining and quantified using a computer assisted image analysis (LEICA Qwin, Rueil‐Malmaison, France). Stained cells were counted over the complete length of the section and reported to the corresponding length of basal epidermis. Inhibition of the tyrosinase/TRP‐1 active complex formation on human melanocytes The normal human melanocytes (NHM) were cultivated in a growth medium (K‐SFM Invitrogen, Cergy, France) supplemented with 50 μg mL ‐1 BPE, 5 ng  mL ‐1 EGF, 10 ng  mL ‐1 bFGF (Invitrogen) and 0.25 μg  mL ‐1 PdBu (Sigma, St Quentin Fallavier, France). NHMs were seeded in 96‐well microplates (Falcon, Franklin Lakes, NJ, U.S.A.) in a proportion of 10 000 cells per well, in 200 μl of growth medium without metabolic activator. Phosphorothioate antisens oligonucleotides (designed from GeneBank accession number X51420 and X06318) were tested at 250 n M , 500  n M or 1 μ M , every day during 5 days. Control sequences and kojic acid (530 μ M ) were used as negative control and positive control respectively. After treatment, cells were rinsed with PBS then 50 μL of lysis buffer 0.5% Triton in PBS were added to the wells and the plate was shaken for 1 hour at 4°C. The reaction was initiated by adding 50 μL of substrate (10 m M L ‐DOPA, Sigma) to each well. DOPAchrome was measured at 450  n M every 2 min, for 1 h and at 37 °C with regular shaking, using an optical density reader (microplate reader 340 ATTC‐SLT‐Labinstrument, Grödig/Salzburg, Austria). The reaction rates were calculated and expressed as 10 ‐4 OD units/min. Results of the DOPA‐oxidase activity were normalized with cell viability measured with XTT assay. Results TRP‐1 expression TRP1 expression was determined by calculating the number of positive cells per millimetres of basal epidermis. TRP1 expression was restricted to melanocytes which were distributed at regular intervals along the base of epidermis ( Fig. 1). No expression of TRP‐1 was observed in melanosomes which had been transferred to neighbouring keratinocytes. We found that solar lentigo skin exhibited 89% TRP‐1 expression more than normal skin ( P  = 0.05). 1Immunolocalization of TRP‐1 in human skin. (a) Representative image of TRP‐1 expression in solar lentigo. (b) Quantification of TRP‐1 on solar lentigo and normal skin for the 10 Japanese women. Statistical differences were evaluated using the paired Student's test. The data is presented as the mean of 10 biopsies.Inhibition of tyrosinase/TRP‐1 active complex formation in human melanocytes The antisense oligonucleotides were studied with regard to their action on melanogenesis inhibition. The assay was based on measurement of the reaction rate for dopa‐oxidase activity. A drop in the reaction rate compared with the control assay shows a decrease in the enzyme activity and consequently a reduction melanin formation. Using antisense oligonucleotides designed for targeting TRP‐1 expression, PKC beta‐I synthesis or both targets in combination, the reaction rate of the dopa‐oxidase activity was studied (Fig. 2). Firstly, we observed a significant decrease in activity (­17%) as a result of antisense targeting tyrosinase/TRP‐1 complex. Secondly, using a sequence specifically targeting PKC beta‐I mRNA, a significant reduction of dopa‐oxidase activity (­13%) was obtained. This result demonstrates the role played by this specific isoform of PKC for tyrosinase activity modulation. Used in combination, these two oligonucleotides induced not only an inhibition of the dopa‐oxidase activity but a stronger inhibiting effect (­36%). Conclusion Considering the important frequency of appearance of the solar lentigo in Japanese skin and the resulting uneven skin color, we have studied the expression of TRP‐1 in solar lentigo and determined a strategy in order to control melanogenesis. We observed that biopsies of solar lentigo from women's Japanese skin exhibited a significant increase in TRP‐1 protein expression, confirming the crucial role exerted by this enzyme in the melanogenesis process. Using the highly specific antisense strategy on normal human melanocytes, we showed that antisens targeting either TRP‐1 expression or PKC beta‐I or both in combination, inhibited dopa‐oxidase activity of human tyrosinase and consequently induced a reduction of melanin synthesis. We report here, the involvement of TRP‐1 in solar lentigo and we demonstrated the regulating and synergistic effect exerted by TRP‐1 and PKC beta‐I on tyrosinase/TRP‐1complex formation and activity. These data led us to consider this overall approach for controlling spot formation and uneven pigmentation. Acknowledgements We thank N. Lachmann, E. Noblesse, Ph. Gasser (Bio EC, Clamart, France) for their helpful assistance and F. Bonté, C. Mahé for their fruitful discussion and support.