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Influence of -melanocyte-stimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes ¹

VICTORIA M. VIRADOR, JACQUELINE MULLER^*, XUFENG WU, ZALFA A. ABDEL-MALEK, ZU-XI YU, VICTOR J. FERRANS, NOBUHIKO KOBAYASHI, KAZUMASA WAKAMATSU, SHOSUKE ITO, JOHN A. HAMMER and VINCENT J. HEARING²

Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, 20892 USA;
^* Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA;
Laboratory of Cell Biology,
Pathology Section, National Heart, Lung and Blood Institute, NIH, Bethesda, Maryland 20892, USA;
Department of Dermatology, University of Cincinnati, Cincinnati, Ohio 45267, USA;
Department of Dermatology, Nara Medical University, Kashihara, Nara 634, Japan; and
Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192, Japan

²Correspondence: Laboratory of Cell Biology, Bldg. 37, Room 1B25, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: hearingv{at}nih.gov

SPECIFIC AIMS

Melanocytes manufacture melanin-loaded organelles called melanosomes and distribute them to neighboring keratinocytes where their presence confers to the skin its color and photoprotective properties. In this study, we used murine melan-a melanocytes and murine SP-1 keratinocytes alone and in coculture to characterize the processes involved in melanosome transfer. We also sought to determine whether two physiological stimulators of mammalian pigmentation—ultraviolet radiation (UV) and -melanocyte-stimulating hormone (MSH)—stimulated melanosome transfer by melanocytes and/or by keratinocytes.

PRINCIPAL FINDINGS

1. Melanosome transfer in cocultures of melanocytes and keratinocytes
We studied melanosome transfer in cocultures of melanocytes and keratinocytes using time-lapse microphotography. During our many hours of observations (not shown), we saw melanocyte dendrite tips frequently and actively contact keratinocytes for several minutes, but they would eventually retract, leaving nothing behind.

Examination of cocultures of melanocytes and keratinocytes by electron microscopy showed several instances of melanosomes inside keratinocytes indicating that melanosome transfer does occur in vitro. Melanocytes and keratinocytes were typically cocultured for at least 4–5 days before examination. We regularly observed phagosomal structures filled with melanosomes inside keratinocytes after uptake of individual melanosomes from purified melanosome preparations. Such clustered distributions of melanosomes within keratinocytes suggest that phagosomal structures are actively formed by keratinocytes during phagocytosis and do not represent the remnants of melanocyte dendrites.

MSH and UV are major physiological signals that modulate skin pigmentation. To assess whether either stimulus would induce melanosome exocytosis, we treated melanocytes with UV or MSH and examined melanin secreted into the culture medium. There were no significant differences in melanin content in media recovered from UV- or from sham-irradiated melanocytes 6 or 24 h after treatment (not shown). In contrast, there was a sharp increase in melanin in the media after 6 h of exposure to 100 nM MSH vs. controls. The MSH-treated values had returned to control levels within 24 h.

The ultrastructure of melanocytes was examined after exposure of cultures to UV, MSH, or ASP (an antagonist of MSH) (Fig. 1 ). After UV exposure, we observed increased accumulation of melanized melanosomes in melanocytes (Fig. 1B ) compared with sham-irradiated controls (Fig. 1A ). In MSH-treated melanocytes, we observed increased blebbing of melanocyte membranes along with many melanosomes in the extracellular spaces (Fig. 1D ). Treatment with ASP induced the rapid appearance of many vesicles in melanocytes (Fig. 1C ).

View larger version (165K): [in this window] [in a new window]   Figure 1. The effects of UV, MSH, and ASP on melanocytes were examined by electron microscopy. A) Control (at 24 h), B) UV-treated (at 24 h) showing more cells containing greater numbers of melanosomes. C) ASP-treated (at 6 h) showing vacuolization and lack of dense melanosomes. D) MSH-treated (at 16 h) showing membrane blebbing and extracellular melanosomes. Consistent results were found between 6 and 24 h. Bar = 1 µm in all panels.

2. Uptake of isolated melanosomes and inert beads by keratinocytes in culture
To study the phagocytic properties of keratinocytes, we used light and electron microscopy to analyze the uptake of purified melanosomes and inert latex fluorescent beads of different sizes (red=0.1 µm or green=1 µm). Uptake of melanosomes or fluorescent beads by keratinocytes was not seen at 30 min (not shown). However, significant uptake of melanosomes and fluorescent beads occurred over 18 h (Fig. 2 ), as demonstrated by the bright perinuclear fluorescence of the beads (Fig. 2A, B ) and by the dense intracellular localization of dark melanosomes (Fig. 2C, D ).

View larger version (125K): [in this window] [in a new window]   Figure 2. Uptake of fluorescent latex beads or purified melanosomes by keratinocytes. Purified melanosomes or different sized fluorescent beads were added in excess to keratinocytes and uptake was followed over time. Significant uptake occurred within 18 h as demonstrated by the bright perinuclear fluorescence in the case of beads (A, B) and by the clear intracellular localization of the dark melanosomes at 24 and 48 h, respectively (C, D). A–C: 20x D: 100x.

We assessed whether size was an important factor in the uptake kinetics by adding mixtures of the two bead sizes to keratinocytes. The 1 µm green beads and the 0.1 µm red beads represent sizes somewhat larger and smaller than melanosomes, which are 0.5 x 0.25 µm. The uptake kinetics of large and small beads into keratinocytes were similar, suggesting that a similar phagocytosis mechanism occurred in both.

We then studied this phenomenon by electron microscopy in order to characterize the subcellular location of internalized melanosomes or latex beads. Keratinocytes were incubated with purified melanosomes or with small or large latex beads (as above). Melanosomes were found inside phagosomes, which contained melanosomes almost exclusively, and as part of a lysosomal structure with mixed contents (not shown). Large beads were found singly in the cytoplasm and were tightly surrounded by phagosomal membranes. In contrast, numerous small beads were found within each lysosome.

From these experiments, we conclude that melanosomes and synthetic beads are ingested by the same phagocytosis mechanism, based on their size, and are taken up in proportion to their relative abundance. This suggests that the phagocytosis process in keratinocytes has no specificity for melanosomes per se and that the distribution of particles (singly or in complexes) within keratinocytes is based simply on their size.

3. Effects of MSH and UV on phagocytic activity of keratinocytes
To determine whether UV and/or MSH influence phagocytosis by keratinocytes, we treated them with those factors and studied their subsequent uptake of beads. Immediately after UV exposure (or sham irradiation), we added fluorescent beads to keratinocytes, harvested them at different times, and measured the uptake of the beads by fluorescence spectroscopy (not shown). A UV dose of 10 mJ/cm² significantly increased bead uptake compared with unirradiated controls, as did 40 mJ/cm², although in the latter case the uptake eventually began to decrease with time, probably due to the higher rate of cell death at that UV dose.

Keratinocytes were pretreated with 100 nM MSH for various times ranging from 30 min to 24 h; the medium was then changed and a suspension of fluorescent latex beads was added. Forty-five min later, the cells were extensively washed and their cytoplasmic contents were extracted and quantitated for phagocytosis. Bead uptake by keratinocytes dramatically increased with time of exposure to MSH for up to 7 h but was not further increased by 24 h of MSH treatment (data not shown).

4. Screening of gene discovery array membranes
In experiments with gene arrays, we obtained independent evidence that melanosome exocytosis can be stimulated by MSH. Many genes reported to be involved in exocytosis were significantly up-regulated in melanocytes treated with MSH for 4 days (data not shown). Concurrently, we exposed those melanocytes to ASP, which antagonizes the MC1R and down-regulates the eumelanin pathway. In most cases, genes up-regulated by MSH were down-regulated by ASP and vice-versa. Some exocytosis-related genes up-regulated by MSH were SNARE proteins. SNARE proteins reside in transport vesicles (vSNAREs) or target membranes (tSNAREs) and are involved in intracellular trafficking of vesicles and membrane fusion of those vesicles. Syntaxin 3A, SNAP-23, and syntaxin-4 were among the tSNAREs up-regulated by MSH, and synaptobrevin was an up-regulated vSNARE.

Other up-regulated genes identified in that screening were ARF2 (ADP-ribosylation factor) and its interacting phospholipase D. ARFs are a family of small GTP binding proteins that are involved in the formation of coated vesicles and vesicular transport, and may have a regulatory role in the actual secretory step. The screening also identified two different 14–3-3 proteins that were up-regulated, proteins that function as adapters in signaling processes and act in the actin cytoskeleton to regulate vesicle targeting.

CONCLUSIONS

The transfer of melanosomes to keratinocytes in the skin as a means for skin tanning and photoprotection has been the subject of many studies. We examined different aspects of this process. Our observations have led us to conclude that an exocytosis/phagocytosis model under paracrine control is the most relevant mechanism for melanosome transfer. Although our time-lapse microscopy system was capable of resolving single melanosomes, we never witnessed direct transfer of melanosomes or dendrite tips under any of the (many) experimental conditions we used. We did, however, see an active effect of keratinocytes on the elongation of melanocyte dendrites. When keratinocyte colonies began to regress, the attached melanocyte membrane was pulled to the point of dendrite extensions, suggesting that strong membrane contact occurs between both cell types. In melanocyte-keratinocyte cocultures 4 days or older, we frequently observed the presence of melanosomes in keratinocytes by electron microscopy.

The effects of physiological signals such as UV and MSH were studied in the context of melanocyte exocytosis and keratinocyte phagocytosis (summarized in Fig. 3 ). In our experiments, exposure to UV resulted in the accumulation of melanosomes in melanocytes and an increased uptake of beads by keratinocytes. Exposure to MSH caused melanocytes to release melanosomes. Exposure of keratinocytes to MSH resulted in their increased uptake of particulate material from the medium.

View larger version (39K): [in this window] [in a new window]   Figure 3. Schematic. Summary of the effects of UV or MSH on melanogenesis and melanosome transfer by melanocytes and keratinocytes. Various proteins identified in the gene array screening that are involved with vesicle targeting to the membrane are indicated (e.g., 14–3-3, ARF2, and vSNARE/tSNARE proteins).

The gene array screening for genes involved in exocytosis provided further evidence that exocytosis-phagocytosis may be the most important mechanism for melanosome transfer. In response to MSH, the expression of many genes associated with intracellular trafficking and exocytosis was up-regulated in melanocytes. The fact that many of those genes were significantly up-regulated in MSH-treated melanocytes and, in most cases, were simultaneously down-regulated in ASP-treated melanocytes strongly suggests that MC1R receptor signaling is important in the modulation of melanosome exocytosis.

It is clear from these results that melanosome uptake is not a specific phenomenon since keratinocytes also phagocytose inert beads and melanosome fractions containing other organelles. We conclude that melanosome transfer to keratinocytes occurs by processes of exocytosis-phagocytosis that are regulated at least in part by local signaling that involves UV and MSH.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0518fje; to cite this article, use FASEB J. (November 29, 2001) 10.1096/fj.01-0518fje

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