Abstract
We have investigated the role of protein kinase C (PKC) in human melanogenesis. The level of PKC activity paralleled the total melanin content in cultured newborn melanocytes. Activation of PKC by treatment with 5 x 10(-7) M phorbol dibutyrate acutely caused a doubling in the activity of tyrosinase, the rate-limiting enzyme in melanogenesis, known to correlate directly with melanin synthesis in these cells. When PKC was depleted to 5-10% of initial levels, there was a 40-50% parallel reduction in tyrosinase activity; and regeneration of PKC activity was associated with the recovery of tyrosinase activity. By Northern blot analysis, the alpha and beta but not the gamma isoforms were detectable in melanocytes. By Western blot analysis, the racially determined pigment level in cultured melanocytes correlated with PKC-beta protein expression. In a pigmented human melanoma line (P-MM4, 20-30 ng melanin/micrograms protein)and its nonpigmented subclone (NP-MM4, undetectable melanin), PKC-alpha mRNA was expressed in both, whereas PKC-beta mRNA was detectable only in P-MM4 cells. Tyrosinase protein level was comparable in both cell lines. When NP-MM4 cell lysate was incubated with melanocyte lysate known to contain PKC-beta, tyrosinase activity per microgram of melanocyte protein in the combined lysate increased, consistent with activation of the previously inactive tyrosinase of NP-MM4 origin. Moreover, NP-MM4 cells transiently transfected with PKC-beta cDNA increased tyrosinase activity from undetectable to detectable levels. These combined data show that PKC-beta regulates human melanogenesis by activating tyrosinase.
Highlights
From the $Departmentof Dermtology, Boston University School of Medicine, Boston,Massachusetts 02118 and lTDepartment of Molecular Biology, Schoolof Medicine, Yokohumu City University, 3-9Fuku-ura, Kanazawa-Ku, Yokohama 236, Japan
Correlation of Total Protein kinase C (PKC) Activity and Melanin ContentTo investigate the possible role of PKC in human melanogenesis, we initially determined whether total melanin content correlates with total activatable PKC
Melanocytes were cultured under standardconditions to near confluency and total PKC activity and melanin content were measured in paired cultures
Summary
Correlation of Total PKC Activity and Melanin ContentTo investigate the possible role of PKC in human melanogenesis, we initially determined whether total melanin content correlates with total activatable PKC. In experiments comparing four donors, PKC activity was 1,450, 1,300, 813, and 613 cpmlyg protein, and melanin content was 95, 65, 25, and 16 ng/Hg protein, respectively, indicatinga moderate correlation in these measurements among individual cultures (Fig. lA).Because these cells are known to be actively melanogenic under the basal conditions [20], if PKC is involved in melanogenesis, part of the PKC should be in an active form. Continued culturingof melanocytes in the presence of PDBu for 2-3 months caused further reduction in total melanin content, from the initial level of 80 f 5 to 20 f 2 ng/pg protein. To determine whether PKC depletion changes growth rate in these cells, growth curves were generated for paired untreated andPDButreated melanocytes for up to 2 weeks (Fig. 2C). The growth rate was unaffected, excluding the possibility that the decreased tyrosinase activity andmelanin content aresecondary to altered growth rate in humanmelanocytes
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