PIGMENT CELL RES 14: 236–242.
2001
Printed in Ireland —all rights reser6ed
Invited Review
Keratinocyte – Melanocyte Interactions During Melanosome Transfer
MIRI SEIBERG
Skin Research Center, J&J CPWW, Skillman, New Jersey
*Address reprint requests to Dr. Miri Seiberg, Skin Research Center, J&J CPWW, 199 Grand6iew Road, Skillman, New Jersey 08558.
E-mail: mseiber@cpcus.jnj.com
Received 14 May 2001; in final form 22 May 2001
The epidermal–melanin unit is composed of one melanocyte
and approximately 36 neighboring keratinocytes, working in
synchrony to produce and distribute melanin. Melanin is
synthesized in melanosomes, transferred to the dendrite tips,
and translocated into keratinocytes, forming caps over the
keratinocyte nuclei. The molecular and cellular mechanisms
involved in melanosome transfer and the keratinocyte –
melanocyte interactions required for this process are not yet
completely understood. Suggested mechanisms of melanosome
transfer include melanosome release and endocytosis, direct
inoculation (‘injection’), keratinocyte–melanocyte membrane
fusion, and phagocytosis. Studies of the keratinocyte receptor
protease-activated receptor-2 (PAR-2) support the phagocyto-
MELANOSOME TRANSFER, EARLY
EM-BASED STUDIES
The epidermal–melanin unit, a functional unit that pro-
duces and distributes melanin, is composed of one
melanocyte and approximately 36 neighboring keratinocytes
(reviewed in (1, 2)). The process of melanogenesis is well
documented and the regulation of pigment production by
melanocytes has been heavily investigated (reviewed in (3)).
Pigment-loaded melanosomes translocate from their site of
origin in the perinuclear cytoplasm towards the melanocyte
dendrite tips using microtubule-based and actin-based mo-
tor proteins (4–6). They are then transferred into the recip-
ient keratinocytes, where they are dispersed into the
cytoplasm. However, the actual transfer of melanosomes
into keratinocytes and the keratinocyte–melanocyte interac-
tions during the transfer process are not well characterized.
Early light and electron microscopy studies documented
the membrane layering and organization during
melanosome transfer, suggesting numerous possible mecha-
Abbre6iations – PAR, protease-activated receptor-2; STI, soybean trypsin inhibitor; IPD, immediate pigment darkening
236
Copyright © Pigment Cell Res 2001
ISSN 0893-5785
sis theory. PAR-2 controls melanosome ingestion and phago-
cytosis by keratinocytes and exerts a regulatory role in skin
pigmentation. Modulation of PAR-2 activity can enhance or
decrease melanosome transfer and affects pigmentation only
when there is keratinocyte – melanocyte contact. Moreover,
PAR-2 is induced by UV irradiation and inhibition of PAR-2
activation results in the prevention of UVB-induced tanning.
The role of PAR-2 in mediating UV-induced responses re-
mains to be elucidated.
Key words: Melanosome transfer, PAR-2, Phagocytosis,
Keratinocyte, Melanocyte
nisms for melanosome transfer. These include the release of
melanosomes into intercellular spaces followed by endocyto-
sis, direct inoculation (‘injection’), keratinocyte –melanocyte
membrane fusion, and phagocytosis. Using time-lapse mi-
croscopy, Okazaki et al. (7) observed dendrites enfolded by
recipient keratinocytes, which are then pinched off to form
a cluster of melanosomes. The internalized dendrites were
decomposed, leaving each melanosome aggregate sur-
rounded by a single keratinocyte membrane. Single large
melanosomes, or complexes of small melanosomes, were
then dispersed from the aggregate into the keratinocyte
cytoplasm, in a skin-type-dependent process defined as cy-
tophagocytosis. Electron microscopy studies of photo-dam-
aged Caucasian facial skin (8) suggested a similar
mechanism, as well as keratinocyte – melanocyte membrane
fusion, and exocytosis of single melanosomes (reviewed in
(5)). However, a mechanistic understanding of the cellular
interactions involved in pigment transfer has not been
described.
Pigment Cell Res. 14, 2001
PHAGOCYTOSIS
Phagocytosis is the cellular internalization of particulate
material of at least 0.5 –1 mm in diameter. Phagocytosis is
receptor-mediated, and receptor activation leads to local
reorganization of the actin cytoskeleton (9). Known phago-
cytic receptors, such as the Fcg receptors, the complement
receptor 3, and the fibronectin receptors (reviewed in (9, 10)),
activate tyrosine and serine–threonine kinases, mobilize
Ca2 + from intracellular storage to the cytoplasm, and acti-
vate arachidonate metabolism (reviewed in (11)). Phagocyto-
sis is usually associated with macrophages, neutrophils, and
monocytes, all ‘professional’ phagocytes functioning to elim-
inate infectious agents, apoptotic cells, and cellular debris
(reviewed in (12)). However, epidermal keratinocytes were
shown to experimentally ingest latex beads (13), demonstrat-
ing their phagocytic ability. The mechanism and regulation
of keratinocyte phagocytosis and its role in epidermal
homeostasis are not yet completely understood.
PROTEASE-ACTIVATED RECEPTOR-2 (PAR-2)
The PAR-2 (14) is a seven-transmembrane G-protein-cou-
pled receptor that is related to but distinct from the thrombin
receptors (TRs, also called PAR-1, PAR-3, and PAR-4
(15–17)). Cleavage of the extracellular domain of PARs by
serine proteases exposes new N-termini, which then act as
tethered ligands (top panel of Fig. 1). Both TRs and PAR-2
are activated by trypsin, but only TRs could be activated by
thrombin (14, 18) and only PAR-2 is activated by mast cell
tryptase (19). Using synthetic peptides that correspond to the
newly created N-termini, PARs could also be activated
without receptor cleavage. SLIGRL and SLIGKV, the
mouse and human PAR-2-activating peptides, are specific for
PAR-2 activation and are equipotent in the activation of the
human PAR-2 (20, 21). The cleaved-activated receptors are
physically coupled to their tethered activating ligands. There-
Fig. 1. A schematic representation
of PAR-2 activation and
inhibition and the effects on
keratinocyte phagocytosis and
melanosome transfer.
Pigment Cell Res. 14, 2001
fore, efficient mechanisms for signal termination were devel-
oped, including cleavage of the tethered ligand, receptor
phosphorylation and uncoupling from G-proteins, and endo-
cytosis and degradation of the activated receptors (11, 22).
The structure–function relationship between PAR-2 and its
peptide antagonists is not fully understood. Reduction of
peptide length, chemical modifications, and amino acid sub-
stitution studies yield different results in different biological
systems, suggesting tissue-specific PAR-2 subtypes (23). The
effect of antagonist binding on receptor structure has not
been studied, and the earliest events documented upon all
PARs activation are Ca2 + mobilization and IP3 hydrolysis
(reviewed in (22, 23)). PARs are expressed in multiple types
of cells and tissues and are involved in growth and develop-
ment, mitogenesis, inflammatory response regulation, malig-
nant transformation, vascular tonus, and blood pressure
regulation. PAR-2 is expressed in keratinocytes (24, 25), but
not in melanocytes (26).
MELANOSOME TRANSFER IN EPIDERMAL
EQUIVALENTS
The experimental reconstruction of the epidermal melanin
unit in vitro was pivotal to melanosome transfer studies.
Using the air–liquid interface system (27), differentiated
keratinocytes were grown in the presence of melanocytes,
enabling scientists to combine microscopy with cellular and
molecular studies. Using three-dimensional keratinocyte–
melanocyte cultures, Herlyn and Shih (28) suggested that
pigment donation occurs through the uptake of dendrite
fragments and phagocytosis of individual melanosomes, the-
orizing that keratinocyte-produced factors regulate this pro-
cess (29). Bessou et al. documented an increase in
melanosome transfer following UVB irradiation of reconsti-
tuted epidermal cultures (30). Melanosomes, either individu-
ally or in clumps, were demonstrated both in the basal layers
237
Fig. 2. PAR-2 modulation affects pigmentation. (a) – (c) Keratinocyte – melanocyte monolayer co-cultures were treated with SLIGRL (10
mM) or with STI (0.01%) for 3 days and stained with DOPA on the fourth day. (d)– (f) HaCaT keratinocytes were treated as indicated above,
followed by incubation with isolated melanosomes, extensive washing, and Fontana & Masson (F&M) staining. (g) – (h) Lightly pigmented
human skins grafted on SCID mice were treated with vehicle (g) or SLIGRL (50 mM, h) for 8 or 9 weeks. F&M-stained histological sections
demonstrate increased pigment deposition and enhanced cap formation following SLIGRL treatment. Bar= 10 mm. (i)– (j) Heavily
pigmented human skins grafted on SCID mice were treated with vehicle (j) or STI (0.1%, i) for 8 or 9 weeks. F&M-stained histological
sections demonstrate reduced pigment deposition following STI treatment.

and in the stratum corneum of these skin equivalents.
Epidermal equivalents of different skin photo-types and
chimeras were introduced next and were also generated from
outer root sheath cells combined with melanocytes (31, 32).
The in vitro formation of nuclear melanin caps was also
reported in epidermal equivalents, completing the process of
skin pigmentation in the reconstituted system (33). To-
gether, these studies demonstrate melanosome synthesis,
transport to keratinocytes, capping of keratinocyte nuclei,
and tanning in epidermal equivalents, enabling the use of
such systems for mechanistic studies.
PAR-2 AFFECTS PIGMENTATION
Studies of skin homeostasis demonstrate an important bal-
ance between serine proteases and their inhibitors, at both
structural and signaling levels (24, 34, 35). In the search for
potential interactions between G-protein-coupled signaling
pathways and serine proteases in the skin, PARs were
evaluated for their possible effect on pigment modulation.
PAR-2 was found to regulate pigmentation (Fig. 2a –c) by
affecting keratinocyte phagocytosis (Fig. 1 (26, 36– 38)).
PAR-2 activation increases the ability of keratinocytes to
ingest melanosomes (Fig. 2d–f), in a process that requires
keratinocyte–melanocyte contact. PAR-2 activation leads to
skin darkening, and inhibition of PAR-2 activation by serine
protease inhibitors reduces pigment transfer and leads to
depigmentation (Fig. 2g– j).
Epidermal equivalents containing melanocytes respond to
PAR-2 activation by darkening, similar to their response
following UVB irradiation (26). Trypsin inhibitors, known

238 Pigment Cell Res. 14, 2001

to inhibit PAR-2 cleavage [e.g., soybean trypsin inhibitor
(STI) (38)], completely inhibited the UVB-induced pigmen-
tation of these equivalents (26). Keratinocyte–melanocyte
contact was required for the PAR-2-mediated pigmentary
effects. Melanocytes alone did not respond to PAR-2 modu-
lation with pigmentary changes. Cultured monolayer
melanocytes grown together with, but with not in contact
with, epidermal equivalents (containing no melanocytes)
were also unaffected by PAR-2 modulation (26). Only two-
and three-dimensional keratinocyte–melanocyte co-cultures
treated with the PAR-2-activating peptide SLIGRL or with
STI showed increased or decreased pigment deposition,
respectively. The finding that PAR-2 is expressed by kerati-
nocytes, but not by melanocytes (26), could explain the
requirement for a keratinocyte –melanocyte contact for the
PAR-2-induced pigmentary effects.
To examine whether modulation of pigmentation by
PAR-2 could be reproduced in vivo, studies were performed
using pigmented Yucatan swine and human skins trans-
planted onto immuno-suppressed mice (see human skin data
in Fig. 2g –j). Both experimental models showed a slight, but
visible, increase in pigmentation following SLIGRL treat-
ment, and an increase in pigment deposition was docu-
mented histologically (Fig. 2g,h (26, 35, 37)). Treatment
with STI resulted in significant depigmentation, and reduced
pigment deposition was observed histologically (Fig. 2i,j (26,
35, 37)). The PAR-2-mediated pigmentary effects were re-
versible (35), suggesting that a process was being modulated,
rather than being permanently changed.
PAR-2 AFFECTS MELANOSOME TRANSFER
VIA KERATINOCYTE PHAGOCYTOSIS
Keratinocytes incubated with powdered melanin or with
isolated melanosomes increased melanosome ingesting fol-
lowing PAR-2 activation. Inhibition of PAR-2 activation by
STI resulted in the opposite effect, suggesting that PAR-2
may affect melanosome ingestion by epidermal kerati-
nocytes (Fig. 2d – f (36)). The role of PAR-2 modulation on
keratinocyte phagocytosis was demonstrated using fluores-
cent-labeled E. coli K-12 bioparticles and fluorescent micro-
spheres. PAR-2 activation induced a dose-dependent,
statistically significant increase in phagocytosis, while STI
reduced keratinocyte ingestion of microspheres or E. coli
particles (37). Similar results were obtained using
macrophages and fibroblasts (37), suggesting a novel role
for PAR-2 in mediating phagocytosis. It is interesting to
note that both PAR-2 and the known phagocytic receptors
share signaling pathways that affect intracellular Ca2 + mo-
bilization (11, 22). The PAR-2-induced phagocytosis is cy-
toskeleton-mediated, and is inhibited by cytochalasin B and
colchicine (37). PAR-2 activation increased actin polymer-
ization at the core region proximal to the plasma membrane,
and STI led to the opposite effect while cytoskeletal protein
levels remain unchanged (37). Electron microscopy studies
showed increased number, and longer and thinner cell pro-
jections (podia), in SLIGRL-treated keratinocytes, while
STI-treated cells showed reduced number and shorter length
podia, relative to untreated controls. In culture, kerati-
nocyte – keratinocyte podia contact was markedly affected
Pigment Cell Res. 14, 2001
by modulation of PAR-2 activity (37, 38), suggesting that
PAR-2 affects cell surface changes having an active role in
keratinocyte phagocytosis.
Melanocytes within epidermal equivalents treated with
trypsin inhibitors contained increased number of
melanosomes, which were less mature relative to untreated
controls (36). Higher number of dendrites with mature
melanosomes was documented within the treated kerati-
nocytes, relative to untreated controls. These data suggest
abnormal melanosome formation and slow or impaired
melanosome transfer into treated keratinocytes. When Yu-
catan swine skins were treated with trypsin inhibitors, aber-
rant melanosome dynamics were detected. Smaller and less
pigmented melanosomes were detected within the treated
epidermal keratinocytes, and the distribution of
melanosomes within the treated skin was abnormal (26).
UVB IRRADIATION INDUCES PAR-2
ACTIVATION
Phagocytosis is associated with protease secretion and acti-
vation (39, 40). PAR-2 activation was found to increase
serine protease secretion (37), which could provide a positive
feedback mechanism for its own activation. Interestingly,
UVB irradiation induced a similar effect. Keratinocytes
treated with increasing doses of UVB irradiation were found
to secrete protease activity, which cleaved a peptide com-
prising the PAR-2 cleavage site in a dose-dependent manner
(41). Since the inhibition of PAR-2 activation prevents
UVB-induced pigmentation in vitro (26), the contribution of
the PAR-2 pathway to UV-mediated responses was exam-
ined in vivo. UV-induced tanning of Yucatan swine was
prevented with topical treatments of STI-containing compo-
sitions (Fig. 3 (38)), demonstrating the importance of
melanosome transfer to the tanning response. PAR-2 upreg-
ulation was demonstrated in human skin following UV
irradiation, and the distribution of the PAR-2 protein within
the epidermis was shown to be UV- and skin-type-depen-
dent (41). In cultured chimeras of different photo-type
origin, the keratinocyte photo-type was correlated with the
pattern of melanosome distribution. On the contrary,
melanosome size or photo-type origin of melanocytes was
not associated with the individual versus clustered pattern of
melanosome transfer (42). These data support the relation
between skin type, UV response, and PAR-2 activation.
Studies of PAR-2 expression levels in keratinocytes from
different skin types could further support a role for PAR-2
in skin photo-type responses.
IMMEDIATE PIGMENT DARKENING (IPD)
AND MELANOSOME TRANSFER
IPD is a transitory darkening of the skin observed immedi-
ately following UV exposure. It involves structural changes
in melanocytes and keratinocytes and a chemical modifica-
tion of pre-existing melanin. Proposed mechanisms for IPD
are controversial. They include photo-oxidation of melanin,
changes in cytoskeletal distribution, movement of
melanosomes to melanocyte dendrites, increased melano-
239
Fig. 3. PAR-2 is involved in UVB-mediated pigmentation. F&M-stained sections of UVB and vehicle-treated Yucatan swine skin (b) show
increased pigment deposition and cap formation relative to untreated control (a). UVB treatment following by daily STI treatment (c)
prevented the UVB-induced tanning.
somes transfer, and changes in the melanosome distribution
pattern within keratinocytes (43, 44).
Most studies of IPD employed UVA only. UVA-induced
IPD leads to photo-oxidation, resulting in darkening of
preformed melanin and production of free radicals. UVA-
induced IPD does not induce cytoskeletal changes in
melanocytes, does not affect melanosome transfer, and
could not be blocked by cytoskeletal disruptive agents (43).
UVA-induced IPD results in no photo-protection, and its
biological role and evolutionary value have been questioned.
Morphological changes in melanocytes and changes in
melanosome transfer and distribution within keratinocytes
were always associated with IPD, but not with UVA-in-
duced IPD. We would like to speculate that UVB-induced
PAR-2 activation, which leads to enhanced melanosome
transfer, might represent an early cutaneous response to
UVB. UVB-induced PAR-2 activation could provide imme-
diate photo-protection by the urgent transfer of available
melanosomes, and could initiate a positive feedback re-
sponse, which would enhance pigment production. The ki-
netics of PAR-2 in UVB-induced immediate responses and
in total spectrum UV-induced IPD remain to be studied.
OTHER KERATINOCYTE MOLECULES
INVOLVED IN MELANOSOME TRANSFER
Keratinocytes produce soluble factors that affect melanocyte
proliferation, dendricity, and melanin synthesis. Condi-
tioned media from UV-exposed keratinocytes is known to
stimulate tyrosinase activity and melanogenesis. However,
keratinocyte factors involved directly in melanosome trans-
fer have not been described in detail.
The trafficking of proteins between intracellular compart-
ments and their delivery to target organelles involves the
docking of secretory granules on the plasma membrane and
the subsequent fusion and release of granule contents. The
Rab–SNARE hypothesis provides a mechanism for the
specific docking and fusion of transport vesicles with their
target membranes (reviewed in (45–47)). A similar mecha-
nism has been implicated in intramelanocyte melanosome
240
transport, and Rab and SNARE proteins were identified on
melanosome membranes (for a recent review of melanosome
transport see (48)). A simple extension of the Rab –SNARE
hypothesis could explain other cellular processes, including
a fusion event between melanosome and keratinocyte mem-
branes. In search for keratinocyte–melanocyte membrane
contact molecules, the possible involvement of Rab and
SNARE proteins in both melanosome transport and trans-
fer has been suggested (49).
Lectins are carbohydrate-binding proteins involved in a
variety of recognition processes. Small alterations in essen-
tially the same tertiary structure result in a high level of
lectin specificity. Cell surface carbohydrates and lectins play
a critical role in cell –cell interactions, in cell adhesion
mechanisms, and in sorting receptors through secretory
pathways (reviewed in (50–52)). Similar to their trafficking
function in the secretory pathway, a role for lectins and
glycoproteins in melanosome transfer has been suggested
(53). The addition of selected lectins to keratinocyte –
melanocyte cultures inhibited melanosome transfer, possibly
by competition with membrane glycoproteins. However, the
possible inhibitory effect of lectins on other cell parameters
remains to be investigated (53).
CONCLUSIONS AND FUTURE PERSPECTIVES
Melanin synthesis within melanosomes and melanosome
distribution within the epidermis determine skin pigmenta-
tion. The distribution of melanin within the epidermal–
melanin unit is regulated, in part, by the keratinocyte
receptor PAR-2. PAR-2 affects melanosome ingestion, and
modulation of PAR-2 activation affects melanosome trans-
fer, contributing to the regulation of skin pigmentation
(Figs 1 and 2). PAR-2 activation induces keratinocyte
phagocytosis, which affects cell podia morphology and cy-
toskeleton reorganization. In vivo, PAR-2 activation results
in increased melanin deposition and enhanced keratinocyte
cap formation. Inhibition of PAR-2 activation in vivo leads
to aberrant melanosome processing, abnormal melanosome
dynamics, and atypical melanosome distribution, resulting
Pigment Cell Res. 14, 2001
in depigmentation. UVB induces PAR-2 expression and
activation, and inhibition of PAR-2 activation prevents
UVB-induced pigmentation in vitro and in swine skin (Fig.
3). Studies are in progress to better understand the role of
PAR-2 in UV-mediated skin darkening. The possible in-
volvement of PAR-2 in human skin-type responsiveness
remains to be elucidated.
Over the past few years, significant advances have been
made towards the understanding of melanosome transfer.
This review focused on the understanding of normal
melanosome transfer in mammalian skin. Emphasis was put
on keratinocyte –melanocyte interactions and on the regula-
tory role of the transfer process in skin pigmentation and
tanning. Future studies are likely to focus on the identifica-
tion of key molecules involved in the dendrite–keratinocyte
interaction, the ‘glue’ molecules that enable keratinocyte
phagocytosis, and their regulation. Further studies should
also aim for a better understanding of keratinocyte phago-
cytosis, the regulation of PAR-2 expression and signaling in
keratinocytes, the natural activators of PAR-2 in skin and
their regulation, and the termination of PAR-2 signaling.
The combination of electronics, optics, and software with
molecular and cellular biology techniques should enable the
analysis of candidate genes and mutations for their
melanosome transfer ability in real time. Better understand-
ing of the molecular mechanisms of melanosome transfer
could result in better control of human skin color.
Acknowledgements – The author thanks all colleagues who have
contributed to these studies. Special thanks to Drs Magdalena
Eisinger and Stanley Shapiro for their thoughts and enthusiasm
throughout the PAR-2 project.
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