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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 3, 2003 as doi:10.1096/fj.02-1059fje. |
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Institute of Cell Biology and Bonner Forum Biomedizin, University of Bonn, 53121 Bonn, Germany;
* Marienhospital, 50321 Brühl, Germany; and
Fondazione Banca degli Occhi del Veneto, 30171 Venezia-Mestre, Italy
2Correspondence: Institute of Cell Biology, University of Bonn, Ulrich-Haberland-Str. 61a, 53121 Bonn, Germany. E-mail: Herzog{at}uni-bonn.de.
SPECIFIC AIMS
The ß-amyloid precursor protein (APP) is strongly expressed in the basal epidermal cell layer where its soluble amino-terminal ectodomain (sAPP) is known to exert growth and motility promoting functions in the epidermis. Using whole-mount preparations of isolated human epidermis, we detected a small population of basal cells that expressed exceptionally high levels of APP. We sought to identify these cells and to study their ability to release sAPP. Once identified, APP might serve as a marker protein of these cells. Results from these studies would pave the way to investigating physiological effects of sAPP on these cells in culture. The expected results would extend our knowledge on protein expression in epidermal cells in general and on the regulatory role of sAPP in the epidermis in particular.
PRINCIPAL FINDINGS
1. High APP expression in epidermal melanocytes and melanoma cells
APP as a marker protein
In whole-mount preparations of isolated human epidermis, cells with exceptionally high APP expression levels showed sufficient contrast to distinguish them from surrounding keratinocytes (Fig. 1
).These cells differed morphologically from keratinocytes by their dendritic shape (Fig. 1A
). This arborization is in the epidermis known only from Langerhans cells and melanocytes. By colocalization of APP with tyrosinase (Fig. 1B
), a key enzyme in melanogenesis, and by their distribution in the epidermis, identical to that of epidermal cells detected by DOPA staining (Fig. 1C
), the dendritic cells were identified as melanocytes. In whole-mount preparations of the epidermis, APP can therefore be used as a marker protein for immunocytochemical visualization of melanocytes and melanoma cells.
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In cell culture we observed that normal human epidermal melanocytes (melanocytes) express APP and secrete sAPP into the surrounding media. For precise quantitation of APP expression, two cell lines [cultured cells of the melanoma cell line A375 (melanoma cells) and HaCaT cells] were biosynthetically radiolabeled, lysed, and subjected to immunoprecipitation. Melanoma cells were included in this study because they are comparable to melanocytes in numerous features. Analyses showed that the characteristic mature (
130 kDa) and immature (110 kDa) forms were both detectable in melanoma cells. APP expression in melanoma cells was fourfold as high as in HaCaT cells.
APP isoforms in melanocytes and melanoma cells
Expression of APP isoforms in melanocytes and melanoma cells was determined by RT-PCR using primers flanking the differentially spliced exons and compared with normal human keratinocytes (keratinocytes), HaCaT cells, and cells of the neuroblastoma cell line SH-SY5Y (neuroblastoma cells). In all cell types, APP isoforms 695, 751, and 770 were detected. However, all epidermal cells, including melanocytes and melanoma cells, expressed predominantly isoforms 751 and 770 and (in contrast to neuroblastoma cells) only low levels of APP 695.
Cellular localization of APP
For details on the localization of APP, we used single cells in culture. Immunocytochemical studies of melanocytes and melanoma cells showed that APP is mainly localized in a reticulum within the perinuclear region and in granules at the tips of dendrites. Colocalization with tyrosinase indicated the expression of APP in premelanosomes, whereas colocalization with protein disulfide isomerase identified the reticular structure as part of the endoplasmic reticulum. APP-containing granules at the tips of dendrites that showed only low tyrosinase activity were identified by their localization and by phase contrast as mature melanosomes. APP in melanosomes was restricted to the melanosomal membranes where APP might be involved in the kinesin-mediated transport of melanosomes along microtubules.
2. sAPP secretion by melanocytes and melanoma cells
sAPP is the amino-terminal ectodomain of APP released by the proteolytic activity of 
-secretase. The question arose whether the high expression levels of APP in melanocytes and melanoma cells result in high rates of sAPP secretion. This could be analyzed only using supernatants of cells in culture. The results obtained by radio-immunoprecipitation revealed that the level of sAPP secretion in melanoma cells was indeed two- to threefold above that of HaCaT cells. To elucidate the possible biological role of sAPP, we prepared eukaryotic recombinant sAPP of all three APP isoforms (sAPPrec), which was applied to melanocytes and melanoma cells cultured in excess medium.
3. The biological role of sAPP in melanocytes and melanoma cells
sAPP as a regulator of lamellipodia dynamics
Melanocytes in situ usually reside in the basal epidermal layer and form multiple long dendritic processes by which melanosomes are transported from the perinuclear region to dendritic tips and transferred to keratinocytes, thereby providing photoprotection of the epidermis. By performing live cell imaging using confocal laser scanning microscopy, we quantified lamellipodia dynamics at the tips of dendrites of melanocytes (data not shown) and melanoma cells (Fig. 2
). Pictures taken at 0, 2, and 5 min illustrate the lamellipodia activity (Fig. 2A
, small images). Using a stroboscopic computer-assisted motility assay we analyzed lamellipodia activity over 5 min. A time space plot based on 150 pictures with an interval of 2 s created a stroboscopic image (Fig. 2B
) that allowed simultaneous analysis of lamellipodia protrusion velocity (v=dx/dt) and lamellipodia frequency. Treatment with sAPPrec (10 nM) or -melanocyte-stimulating hormone (-MSH; 50 nM) strongly increased the velocity (Fig. 2C
) and frequency (data not shown) of lamellipodia protrusion. sAPPrec 695 showed the strongest effect and increased lamellipodia protrusion velocity by two- to threefold.
Stimulation of melanin release by sAPP
Increased activity of the dendritic lamella has been shown to coincide with the release of melanin, and -MSH and UVB light appears to raise this activity. Our observations showed that similar to -MSH and UVB radiation (2 mJ/cm2), treatment with sAPPrec 695 stimulates the release of melanin from melanoma cells. The extracellularly accumulating melanin particles were visualized by phase contrast micrographs. Morphometric quantitation of released melanin particles 8 h after treatment showed that sAPPrec 695 exerts a stimulatory effect comparable to that of -MSH, increasing the release of melanin from melanoma cells about twofold. UVB radiation had the strongest effect on melanin release. By transmission electron microscopy, the released melanosomal content was found to consist of highly electron-dense particles due to their composition of melanin. The melanin particles were not surrounded by membranes, supporting the view that their release occurred by exocytosis and suggesting that the melanosomal membrane was inserted into the plasma membrane during this process.
CONCLUSIONS
Here we show the high expression of APP in epidermal melanocytes and melanoma cells and its applicability as a marker protein for this cell type. Particularly in whole-mount preparations of the isolated human epidermis, the high APP expression compared with the surrounding keratinocytes together with the unique dendritic morphology is seen. Results obtained by radio-immunoprecipitation confirmed the high contrast of APP expression between both cell types. However, APP is not a selective melanocyte marker as it is expressed at much higher levels in melanocytes and also found in keratinocytes (Fig. 3
). In this respect, APP shares features with other useful but nonspecific melanocyte markers such as MC1-R, which exhibits an exceptionally high expression level in melanocytes, but is seen in other cell types of the skin such as fibroblasts and endothelial cells.
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We have shown that melanocytes and melanoma cells predominantly express the APP isoforms 751 and 770 and, in contrast to neuronal cells, only low levels of APP 695. Although melanocytes and cells of the central nervous system share a common embryonic origin, melanocytes have adopted an expression pattern similar to that of keratinocytes (Fig. 3
). Melanin transfer to keratinocytes is essential for its role in epidermal photoprotection. Melanocytes constitute, however, only 3% of all epidermal cells, whereby each melanocyte together with the corresponding keratinocytes forms the "epidermal-melanin unit." Hence, the efficiency of melanin transfer depends largely on the regulation of the contacts between keratinocytes and the dendritic tips of melanocytes where mature melanosomes collect. We have shown that melanocytes and melanoma cells are able to secrete sAPP and, together with keratinocytes, contribute to the pool of sAPP in the epidermis. Our results with sAPPrec indicate that, similar to -MSH, sAPP operates as a potent regulator of melanocyte motility and melanin release (Fig. 3
) at the dendritic tips. Both functions may be part of the same operational step: the release of melanin particles results in uptake by keratinocytes only if both cell types are in close contact. Lamellipodia dynamics at the tips of dendrites and its stimulation by sAPP (Fig. 3
) may bring about the contacts between both cell types and facilitate the release of melanin.
Marker proteins for melanocytes and melanoma cells and factors involved in the regulation of their specific function are of great interest. We conclude that APP can be considered a marker protein and that its amino-terminally located ectodomain belongs to the family of epidermal growth factors stimulating melanocyte specific functions. The mode of action of sAPP in the epidermis (Fig. 3
) is comparable to other growth factors in the epidermal-melanin unit: paracrine when sAPP is derived from keratinocytes and autocrine if released by melanocytes.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1059fje; doi: 10.1096/fj.02-1059fje 
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