The synthesis and processing of melanosomes, the pigmented organelles of the follicular and interfollicular epidermis, is of major interest in the field of cutaneous biology. Additionally, clarification of the biological and cellular processes of cutaneous pigmentation has several therapeutic and cosmetic based applications, including the alleviation of skin hyperpigmentation. Modification of skin complexion coloration has traditionally been accomplished by inhibition of the rate limiting enzyme of melanogenesis, tyrosinase, or attenuation of melanosome transfer from melanocytes to keratinocytes. The post transfer modification of pigmented melanosomes, the main focus of this dissertation project, provides an attractive and distinct avenue of modulating skin pigmentation. There is currently limited information on how epidermal keratinocyte process recipient melanosomes during terminal differentiation. Furthermore, the variability of melanosomal degradation seen between light and dark skin remains to be clearly established. Therefore, we have developed a novel model system to investigate the degradation of isolated melanosomes by cultured human keratinocytes. Fluorescently labeled and isolated melanosomes, cultured in the presence of light and dark skin derived keratinocyte cultures, were assessed for degradation. The extent of degradation has been qualitatively assessed, using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Within 48 hours of melanosome incorporation, indirect immunofluorescence and confocal microscopy images suggest that light derived keratinocytes may have accelerated melanosome degradation compared to dark keratinocytes. This time dependent decrease in fluorescence was then quantitatively analyzed using flow cytometry analysis. Consistent with the results of the confocal analysis, over a 48 hour time frame, light keratinocytes appear to degrade melanosomes more efficiently than the dark skin derived keratinocytes, P=0.039. This methodology offers a novel mechanism to address the differential ability of light and dark keratinocytes to degrade melanosomes.
To further delineate the process of melanosome degradation between light and dark skin, we focused on hydrolytic enzymes that have been implicated in epidermal differentiation and potentially melanosome degradation. To investigate this, we performed preliminary microarray analysis on suprabasal epidermis derived from light and dark skin, by laser capture microscopy (LCM). Data analysis of the microarray experimentation showed over-expression of various hydrolytic enzyme genes in the suprabasal epidermal layers, when comparing light to dark skin. Western blot analysis performed to confirm the expression pattern of hydrolytic enzymes from either light or dark skin derived epidermal lysates, demonstrated that cathepsin L2 was reproducibly upregulated in light skin, P=0.048. In addition, immunofluorescence analysis of cathepsin L2 in light and dark foreskin cryosections confirmed this differential expression and demonstrated that this enzyme was expressed throughout the epidermal layers. Biochemical analysis of cathepsin L2 activity in the two complexion types confirms an elevated enzyme activity in light compared to dark skin complexion samples, 1.75 fold higher activity in light skin compared to dark skin, P=0.03. Taken together these results confirm the differential expression of the acid hydrolase cathepsin L2 in light and dark skin at the gene and protein level. These results may have identified a specific acid hydrolase that may play a role in melanosome degradation and pigment processing.