Journal of Cell Science 105, 179-190 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
179
Expression of integrin receptors and their role in adhesion, spreading and
migration of normal human melanocytes
Giovanna Zambruno1, Pier Carlo Marchisio2, Antonella Melchiori3, Sergio Bondanza3, Ranieri Cancedda3
and Michele De Luca3,*
1Clinica Dermatologica, Università di Modena, 41100, Modena, Italy
2Dipartimento di Scienze Biomediche e Oncologia Umana, Università di
3IST, Istituto Nazionale per la Ricerca sul Cancro, 16132, Genova, Italy
Torino, 10126, Torino, Italy
*Author for correspondence
SUMMARY
Integrin receptors of human melanocytes in vivo and of
melanocytes isolated and cultured from in vitro reconstituted normal human epidermis were investigated.
Melanocytes were studied by high-resolution immunocytochemistry of in situ epidermis and were found to
expose only the integrin subunits a3, a6, av and b1 on
their plasma membrane surface. Instead, cultured
normal melanocytes expressed a3b1, a5b1, a6b1 and
avb3, which were immunoprecipitated from both metabolically and surface-labeled cells. Beta1 integrins were
diffused on the adhesion surface, while avb3 was clustered in focal contacts both in control cells and upon
dendrite induction with phorbol 12-myristate 13-acetate
(PMA). The functional roles of integrins were studied
in vitro by cell adhesion, spreading and migration
assays. The sum of the data indicated that, in normal
human melanocytes: (i) adhesion to defined substrata is
mainly mediated by specific b1 integrins; (ii) spreading
is mainly modulated by avb3; (iii) the b1 and b3 heterodimers cooperate in regulating migration. The in
vitro expression of two integrins (avb3 and a5b1) that
are not exposed in situ, and their role in the spreading
and migratory properties of melanocytes, strongly
suggest that they are involved in regenerating a normally pigmented epidermis during wound healing by
controlling melanocyte spreading and migration over a
provisional matrix. Tumor promoters, such as PMA,
selectively increased the expression of a3b1. We suggest
that this integrin might be involved in melanocyte
migration on the newly formed basement membrane
during wound healing as well as in intercellular recognition of adjacent keratinocytes.
INTRODUCTION
1988a) and suitable for autologous and permanent grafting
onto patients presenting large skin and mucosal defects
(Gallico et al., 1984, 1989; De Luca et al., 1989, 1990a;
Romagnoli et al., 1990). Keratinocytes stimulate
melanocyte growth, regulate the melanocyte-keratinocyte
ratio, melanocyte morphology and melanin synthesis, and
specifically direct the proper spatial and physiological
organization of melanocytes within the basal layer of cultured epidermis (De Luca et al., 1988a,b; Gordon et al.,
1989; Haake and Scott, 1991; De Luca et al., 1993).
While considerable information is available on the
growth requirements and on the regulation of the adhesive
and migratory properties of melanoma cells (for recent
reviews, see Halaban, 1991; Kramer et al., 1991a; Hart et
al., 1991; Cheresh, 1991), the molecular mechanisms
responsible for: (i) the adhesion of normal human
melanocytes to the underlying basement membrane; (ii) the
physical
interaction
between
keratinocytes
and
melanocytes, leading to the formation of the epidermal-
Melanocytes are neural crest-derived cells endowed with
defined morphological and biochemical markers, and are
mainly located within the basal layer of the epidermis.
Melanocytes lie on the epidermal basement membrane
where they closely interact with surrounding basal keratinocytes to form a functional structure defined as the epidermal-melanin unit (Weiss and Greep, 1977). Melanocytes
protect keratinocytes against ultraviolet damage (such as
photocarcinogenesis) through the synthesis of the melanin
pigment and the transfer of melanosomes to adjacent basal
keratinocytes by multiple long dendritic processes (Weiss
and Greep, 1977). Normal human skin keratinocytes and
mucosal epithelial cells generate in vitro a stratified squamous epithelium closely resembling the epithelium in vivo
(Rheinwald and Green, 1975; Green, 1980; Compton et al.,
1989; De Luca et al., 1990a,b; Pellegrini et al., 1992), with
the physiological density of melanocytes (De Luca et al.,
Key words: integrin, melanocyte, melanoma, skin, migration,
adhesion
180
G. Zambruno and others
melanin unit; and (iii) the migration of melanocytes during
the healing of wounds, leading to wound re-pigmentation,
remain largely unknown. This is of particular interest, since
alterations in the adhesive and migratory properties of
normal human melanocytes, together with alterations in keratinocyte-melanocyte recognition phenomena (De Luca et
al., 1993), can lead to several types of skin pathology
including highly malignant melanomas.
Integrins are a super-family of transmembrane heterodimeric (a and b subunits) surface receptors involved
in cell-matrix and cell-cell adhesion (for recent reviews see
Rouslahti, 1991; Hynes, 1992). So far, at least 13 a subunits and 7 b subunits have been isolated and characterized. The combination of individual a and b subunits into
complexes may yield a large number of potential receptors.
Integrins mediate cell adhesion by recognizing specific
amino acid sequences within components of the extracellular matrix and/or specific proteins exposed on the surface
of adjacent cells (Hynes, 1987, 1992; Rouslahti, 1991).
Recently, our laboratories have highlighted the role of
specific integrins in the control of keratinocyte polarity as
well as in epidermal morphogenesis, both in normal skin
and in hyperproliferative skin diseases (De Luca et al.,
1990b; Marchisio et al., 1991; Pellegrini et al., 1992; De
Luca et al., 1992; Savoia et al., 1992).
In this study we investigated the expression of integrin
receptors in normal human melanocytes in vivo, and after
their isolation from in vitro reconstituted human epidermis.
We report that: (i) human melanocytes in intact epidermis
in vivo express the integrin subunits a3, a6, av and b1; (ii)
normal human melanocytes, isolated in culture, express the
heterodimers a3b1, a5b1, a6b1 and avb3; (iii) the b1 integrins mediate melanocyte adhesion to specific substrata,
while avb3 does not play any role even on vitronectin; (iv)
the avb3 integrin plays a crucial role in regulating
melanocyte spreading and migration; (v) exposure of
melanocytes to tumor promoters (such as phorbol 12-myristate 13-acetate; PMA) selectively increases the expression
of the a3b1 heterodimer.
MATERIALS AND METHODS
Antibodies
The rabbit polyclonal antiserum to b3 and the goat antiserum to
b1 have been described (Dejana et al., 1988; Conforti et al., 1989).
The rabbit polyclonal antiserum 5710 to b4 (Kajiji et al., 1989)
and the rabbit polyclonal antiserum to b6 (Sheppard et al., 1990)
were generous gifts from V. Quaranta, Research Institute of
Scripps Clinic, La Jolla, CA. The rabbit polyclonal antiserum to
b5 (Ramaswamy and Hemler, 1990) and the murine monoclonal
antibody (mAb) TS2/7 to a1 (Hemler et al., 1983) were gifts from
M. Hemler, Dana-Farber Cancer Institute, Boston, MA. The rabbit
polyclonal antiserum to a7 (aCD, Song et al., 1992) was a gift
from S. Kaufman, University of Illinois at Urbana-Champaign,
Urbana, IL. Other mAbs, with the investigators that kindly provided them, are as follows: J143, to a3 (Fradet et al., 1984), from
L. J. Old, Memorial Sloan Kettering Cancer Center, New York,
NY; GoH3 to a6 (Sonnenberg et al., 1987), from A. Sonnenberg,
The Netherlands Cancer Institut, Amsterdam, The Netherlands);
BIIG2 to a5, and AIIB2 to b1 (Werb et al., 1989), from C.
Damsky, University of California, San Francisco, CA; HP1/7 to
a4 (Pulido et al., 1991), and TS1/8 to b2 (Sanchez-Madrid et al.,
1983), from F. Sanchez Madrid, Hospital de la Princesa, Madrid,
Spain; LV230 to av (Houghton et al., 1982), from C.E. Klein,
Department of Dermatology, University of Ulm, Germany; 13C2
to av (Horton et al., 1985), from M. Horton, ICRF, London, UK;
LM 609 to avb3 (Cheresh and Harper, 1987), from D. Cheresh,
Research Institute of Scripps Clinic, La Jolla, CA. Other mAbs
where commercially obtained: Gi9 to a2 and SAM 1 to a5
(Immunotech, Marseille, France); 3E1 to b4 (Telios, San Diego,
CA); P1B5 to a3 (Chemicon, Temecula, CA).
Keratinocyte culture
Human epidermal keratinocytes were obtained from neonatal foreskins or from skin biopsies of healthy volunteers and cultivated
on a feeder-layer of lethally irradiated 3T3-J2 cells (a gift from
Prof. Howard Green, Harvard Medical School, Boston, MA) as
described (De Luca et al., 1988b). Briefly, skin biopsies were
minced and trypsinized (0.05% trypsin/0.01% EDTA) at 37°C for
3 h. Cells were collected every 30 min, plated (2.5×104/cm2) on
lethally irradiated 3T3-J2 cells (2.4×104/cm2) and cultured in a
5% CO 2 humidified atmosphere in keratinocyte growth medium:
Dulbecco-Vogt Eagle’s (DMEM) and Ham’s F12 media (3:1 mixture) containing fetal bovine serum (FBS, 10%), insulin (5 mg/ml),
transferrin (5 mg/ml), adenine (0.18 mM), hydrocortisone (0.4
mg/ml), cholera toxin (CT, 0.1 nM), triiodothyronine (2 nM), epidermal growth factor (EGF, 10 ng/ml), glutamine (4 mM), penicillin-streptomycin (50 i.u./ml). Subconfluent primary cultures
were trypsinized, cells were plated in secondary cultures at a density of 4×103 to 1.3 ×104 cells/cm2 and cultured as above. 3T3-J2
cells were cultured in DMEM containing bovine serum (10%),
glutamine (4 mM) and penicillin-streptomycin (50 i.u./ml).
Melanocyte culture
Normal human melanocytes were isolated from in vitro reconstituted epidermis. Confluent cultures (from keratinocytes in primary
or secondary culture) were trypsinized (2-3 days after confluence)
and the cell suspension was plated in the absence of feeder-layer,
at a cell density of 2.5×104/cm2 in melanocyte growth medium
(MGM): E-199 containing FBS (5%), insulin (5 mg/ml), transferrin (5 mg/ml), adenine (0.18 mM), hydrocortisone (0.4 mg/ml),
CT (0.1 nM), triiodothyronine (2 nM), EGF (10 ng/ml), basic
fibroblast growth factor (bFGF, 1 ng/ml), glutamine (4 mM), penicillin-streptomycin (50 i.u./ml). Geneticin (100 to 250 mg/ml) was
added for 2-4 d to avoid human fibroblast overgrowth. Twentyfour hours after seeding, the medium was changed and free-floating cells were removed. After 2-4 d, melanocytes, identified by
their morphology and reactivity to 1,3,4-dihydroxyphenylalanine
(DOPA), were further purified by differential trypsinization:
melanocytes, preferentially detaching after short trypsinization
times (2-3 min), were collected in MGM and plated at a cell density of approximately 5×103/cm2. Subconfluent cultures were passaged 1:3 and cultured as above. After their isolation (100% positivity to DOPA reaction, usually obtained after 2-4 passages),
melanocytes were used in experiments. DOPA reaction was performed as described (De Luca et al., 1988a). PMA (10 ng/ml) was
added to some cultures during the first melanocyte isolation step
and removed thereafter. Melanocytes were used between passages
5 and 10. For immunofluorescence, immunoprecipitation, cell
adhesion and migration assays, and northern analysis, a group of
melanocyte cultures was maintained in MGM and a second group
was treated with PMA (50 ng/ml) for 48 h before performing the
experiments.
Immunoelectron microscopy
Immunoelectron microscopy was performed on normal intact epidermal sheets separated from skin specimens by dispase treatment
Integrins in normal human melanocytes
as previously described (Kanitakis et al., 1992). Briefly, keratomized skin obtained from plastic surgery was floated on 0.5%
dispase (neutral protease grade II, Boehringer-Mannheim,
Mannheim, Germany) for 1 h at 37°C. Epidermal sheets were
separated from the dermis and incubated for 1 h at 4°C with mAbs
to a1, a2, a3, a4, a5, a6, av, avb3, b1, b2, b4 and unrelated
mAbs, and with antisera to a7, b5 and b6 in RPMI 1640 supplemented with 5% heat-inactivated human AB serum and 5% normal
goat serum; after washing, the sheets were incubated with antimouse, anti-rat and anti-rabbit antibodies coupled to 10 nm colloidal gold particles (Amersham International, Bucks, UK) diluted
1:5 for 1 h at 4°C. Thereafter, the specimens were fixed in 2%
glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in an
ethanol series and embedded in Epon. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined under
a Philips EM 400 electron microscope (Centro Interdisciplinare
Grandi Strumenti, Modena, Italy).
Immunofluorescence
Cultured melanocytes were plated onto 24-well Costar plates
(3×104 cells/cm2) containing 1.1 cm2 round glass coverslips. After
2-3 d, coverslip-attached melanocytes were fixed in 3%
paraformaldehyde in phosphate buffered saline (PBS), pH 7.6,
containing 2% sucrose for 5 min at room temperature. Cells were
permeabilized by soaking coverslips for 3-5 min at 0°C in
HEPES/Triton X-100 buffer (20 mM HEPES, pH 7.4, 300 mM
sucrose, 50 mM NaCl, 3 mM MgCl2 and 0.5% Triton X-100).
Indirect immunofluorescence on cell cultures was performed as
previously reported (Marchisio et al., 1991). Briefly, the primary
antibody (10-30 mg/ml) was layered on fixed and permeabilized
cells and incubated in a humid chamber for 30 min. After rinsing
in PBS-0.2% BSA, coverslips or tissue sections were incubated
in the appropriate rhodamine-tagged secondary antibody
(Dakopatts, Copenhagen, Denmark) for 30 min at 37°C in the
presence of 2 mg/ml of fluorescein-labeled phalloidin (F-PHD;
Sigma Chemical Co., St. Louis, MO). Coverslips were mounted
in Mowiol (Hoechst AG, Frankfurt-Main, FRG) and observed with
a Zeiss Axiophot photomicroscope equipped with epifluorescence
lamp and usually with Planapochromatic oil immersion lenses.
Fluorescence images were recorded on Kodak T-Max 400 films
exposed at 1000 ISO and developed in T-Max Developer for 10
min at 20°C.
Immunoprecipitations
Immunoprecipitations were carried out as previously decribed (De
Luca et al., 1990b; Pellegrini et al., 1992). Briefly, melanocytes
were incubated for 15-20 h in methionine- and cysteine-free culture media in the presence of 100 mCi/ml of [35S]methionine and
100 mCi/ml of [35S]cysteine (Amersham). After labeling, cells
were detached with 10 mM EDTA, 0.02% KCl in PBS, pH 7.4,
and washed twice in PBS containing 1 mM CaCl2 and 1 mM
MgCl2. Cell surface-labeling was carried out with cells in suspension. Cells were detached with 15 mM EDTA, as described
above. After washing in PBS, cells were resuspended (10×106
cells/ml) in PBS, pH 7.4. Iodination was carried out for 15 min
on ice by addition of 1 mCi of 125I (Amersham), 0.25 mg of lactoperoxidase, 0.001% H2O2. Cells were then washed 4 times in
PBS containing 5 mM KI.
Metabolically and surface radiolabeled cells were lysed for 30
min on ice in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1%
deoxycholate, 1% Triton X-100, 0.1% SDS, 0.2% sodium azide),
pH 8.5, containing 4 mM phenylmethylsulfonyl fluoride, 0.2
TIU/ml aprotinin and 10 mg/ml leupeptin. Lysates were then centrifuged and cleared by the addition of one-tenth the volume of
Pansorbin (Calbiochem, San Diego, CA). Immunoprecipitations
were carried out by overnight incubations of the immunoadsor-
181
bents (antibodies adsorbed onto Protein A-Sepharose, Pharmacia,
Uppsala, Sweden) with samples of cell lysates, followed by extensive washing, elution by boiling in Laemmli sample buffer and
reduction with 10 mM dithiothreitol (Pellegrini et al., 1992). The
procedure was strictly performed at 4°C. Samples were then analyzed by SDS-PAGE on 6% polyacrylamide slab gels, followed
by autoradiography on Hyperfilm b-max (Amersham). Proteinbound radioactivity in cell lysates was counted and equivalent
amounts of radioactivity were immunoprecipitated.
Northern blot analysis
The a3 human cDNA probe (clone 3.24) was a gift from Dr M.
Hemler, Dana-Farber Cancer Institute, Boston, MA.
Keratinocyte cultures were used as positive internal control.
Total cellular RNA was isolated by lysing the cells with 4.2 M
guanidine thiocyanate followed by cesium chloride gradient centrifugation as described (Di Marco et al., 1991). A 20 mg sample
of total RNA was size-fractionated through a 1% agarose gel containing formaldehyde, and transferred to a nylon membrane (GeneScreen Plus, Du Pont-New England Nuclear, Bad Homburg,
FRG). After immobilization by short-wave UV exposure, blots
were prehybridized at 42°C for 3 h in 50 % deionized formamide,
0.75 M NaCl, 25 mM sodium phosphate, 5 mM EDTA, 0.2 mg/ml
salmon sperm DNA, 0.5% SDS. Hybridization buffer was identical to the above buffer with the addition of the 32P-labeled a3
probe (2×106 c.p.m./ml) and 10% dextran sulfate. Filters were
washed at 65°C for 30 min in 15 mM NaCl, 1 mM sodium phosphate, 1 mM EDTA, 0.1% SDS and autoradiographed on X-ray
films (Hyperfilm MP, Amersham) with intensifying screens at
−70°C. Equal amounts of RNA were loaded, as assessed by ethidium bromide staining and hybridization with a specific cyclophilin
cDNA.
Adhesion assays and cell spreading
Adhesion assays were performed as previously described (De
Luca et al., 1990b). Twenty-four-well plates were coated with
mouse laminin (Gibco, Gaithersburg, MD; 10 mg/ml in PBS for
1 h at room temperature), human plasma vitronectin (Sigma; 5
mg/ml in PBS for 2 h at 37°C), and human plasma fibronectin (a
gift from L. Zardi, IST, Genova, Italy; 10 mg/ml for 1 h at room
temperature). Melanocytes were plated (1×105 cells/well) in MGM
medium without FBS. Cell were allowed to adhere at 37°C for
varying periods of time and then the non-adherent cells were
removed by washing the wells with PBS. Adherent cells were
fixed, stained with 0.5% crystal violet, washed and dried. The dye
was eluted with 50% EtOH/0.1 M sodium citrate, pH 4.2.
Absorbance was read in a Titertek Multiscan (Flow Laboratories)
at 540 nm. In short-term assays, non-specific adhesion, evaluated
either on glass or on bovine serum albumin (0.1%), was negligible.
For inhibition assays, melanocytes were preincubated for 1 h at
37°C with gentle shaking, with: goat antiserum to b1 and mAbs
LM 609 to avb3, P1B5 to a3, BIIG2 to a5, GoH3 to a6, 13C2
to av, control normal goat serum, or unrelated antibodies. Cells
were then plated (1×105 cells/well) in MGM medium without FBS
in the presence of the appropriate antibodies (1:50 dilutions for
the polyclonal antisera and ascites fluid; 1:3 dilutions for the mAbs
supernatants, unless otherwise indicated). After 30 to 120 min at
37°C cells were fixed and stained as above.
Cell spreading was evaluated (2 h after plating) in the same
conditions as for adhesion assays. Random triplicate fields containing at least 100 cells were visually evaluated and photographed.
Cell migration assays
Cell migration assays were carried out in Boyden chambers as
182
G. Zambruno and others
previously described (Albini et al., 1987). Briefly, melanocytes
were resuspended in DMEM without serum and preincubated
with the same antibodies as for adhesions assays, for 2 h at 37°C
with gentle shaking. They were then added (3×105 cells/ml) to
the upper compartment of the Boyden chamber and the lower
compartment was filled with medium containing a chemoattractant: laminin (50 mg/ml), fibronectin (25 µg/ml), vitronectin (8
µg/ml) and control medium (DMEM without serum, in the presence of 0.1% bovine serum albumin). The chemoattractants were
used at concentrations exerting maximal chemotactic activity, as
evaluated in preliminary experiments. The two compartments of
the Boyden chambers were separated by a polycarbonate filter (8
mm pore size, Nucleopore, Concorezzo, Italy) coated with gelatin
(5 µg/ml, Sigma). Melanocytes were allowed to migrate for 12
h at 37°C in a humidified atmosphere containing 5% CO2. Cells
on the upper side of the filter were removed mechanically; cells
on the lower surface of the filter were fixed in ethanol, stained
with toluidine blue, and 10 random fields per filter were counted
at ×160 with a microscope. Each assay was carried out in triplicate and repeated at least three times. In preliminary experiments,
in order to check the ability of the cells (alone or preincubated
with the various antibodies) to adhere to the filters, the upper
side of the filter was fixed, stained and observed under a microscope.
RESULTS
Immunoelectron microscopy
Intact epidermal sheets were obtained from skin specimens
by treatment with the neutral protease dispase II and immunoelectron microscopy was performed as described in
Materials and Methods. Melanocytes were easily identified
by their basal location, their characteristic dendritic morphology (Fig. 1A), and the presence of immature and
mature melanosomes within their cytoplasm (see also
DeLuca et al., 1988a). Epidermal sheets were stained with
mAbs to a1, a2, a3, a4, a5, a6, av, avb3, b1, b2, b4 integrin subunits. Positive melanocytes within the epidermis
were identified by the presence of gold particles (at least
three gold particles on their membrane; see Matutes and
Catowsky, 1982) scattered along the cell membrane. As
shown in Fig. 1, melanocytes were b1 (a, inset), a3 (b, at
arrows), av (c, at arrows) and a6 (d, at arrows) positive.
Eight to thirty gold particles were regularly present on the
cell membrane of each melanocyte observed. Gold particles were uniformely distributed along the melanocyte cell
surface and were also observed along dendritic processes.
When epidermal sheets were labeled with anti-a1 mAb,
very few gold particles were observed on only some
melanocytes (not shown). With all the other mAbs, including the mAb against the b3 subunit, the ‘official’ av partner, we did not observe any gold particles. Immunoelectron
microscopy performed with antisera against a7, b5 and b6
gave negative results as well (not shown). All the antibodies stained the appropriate positive controls (a2, b4 and b5
on keratinocytes; a4, b2 and b3 in tonsil sections; a5 in
dermal sections), while unrelated mAbs were negative.
With mAbs to b4 and a2, a strong labeling of basal keratinocytes was observed, while no gold particles were
detected on melanocytes, thus providing a negative internal control (not shown). Identical results were obtained in
epidermal sheets from three different skin specimens.
Integrin expression in cultured melanocytes
Immunoprecipitations were performed on four different
normal human melanocyte strains isolated and cultured
from in vitro reconstituted human epidermis. Since identical results were obtained in all strains, only immunoprecipitations from strain MP31 are shown (Figs 2-4). Cells
were metabolically labeled and immunoprecipitated (see
Materials and Methods) with antisera to b1, b3, b4, b5, b6
and a7 integrins and with mAbs to the b2 and a1-6 subunits. As shown in Fig. 2, and in contrast with the in vivo
data, both b1 and b3 integrins were immunoprecipitated.
The other b chains, i.e. b2 and b4 to b6 were absent; the
function of these antibodies was tested by immunoprecipitation on cells expressing specific integrins (b2 in U-937
cells; b4 and b5 in normal human keratinocytes; b6 in FG
cells). Note that in cells that were preincubated with PMA,
there was a slight increase in the intensity of the bands in
the a (s) subunit region (at arrows), both in non-reducing
(NR) and in reducing (R) conditions (in R conditions the
a (s) co-migrate with the associated b1 subunit). Metabolically labeled cells were then immunoprecipitated with
mAbs to several a subunits. As shown in Fig. 3, the following heterodimers were immunoprecipitated: a3b1, a5b1,
a6b1 and avb3. Antibodies to other a subunits, including
the melanoma-associated a2, a4 and a7, gave negative
results (not shown). Melanocytes cultured in vitro have
been reported to express appreciable amounts of the a1b1
heterodimer (Kramer et al., 1991a). Limited to this integrin,
we performed immunoprecipitations on seven different cell
strains at different cell passages. In our hands, we noticed
the expression of a very low amount of a1b1 in only two
strains, both at the tenth passage (not shown). Melanocytes,
as well as melanoma cells, represent an heterogeneous cell
population (Kramer et al., 1991a; De Luca et al., 1993).
The variable expression of the a1b1 complex by a selective subset of melanocytes (see also immunoelectron
microscopy data), might thus explain the discrepancy of
results concerning the expression of this integrin. To investigate the cell surface expression of the heterodimers
immunoprecipitated from metabolically labeled cells, isolated melanocytes were surface radio-iodinated and
immunoprecipitated as described in Materials and Methods.
As shown in Fig. 4, integrins a3b1, a5b1, a6b1 and avb3
were all exposed on the melanocyte cell membrane.
It should be noted that: (i) the b3 subunit, recently associated with melanoma cell invasion and migration (Felding-Habermann et al., 1992; Leavesley et al., 1992; Seftor
et al., 1992), was indeed expressed by normal human
melanocytes in culture; (ii) av was associated only with b3
(see the av lane in NR conditions of Fig. 3, and the av lane
of Fig. 4); (iii) both in NR and R conditions (Fig. 3), there
was a selective and strong increase in the a3b1 integrin in
cells following pretreatment with PMA. The a3 lane of Fig.
3 (NR) indeed shows a large amount of the a3 precursor
(the lower band of the a3 doublet), suggesting an increase
in the a3 transcription rate. Immunoprecipitation of surface
radio-iodinated cells, showed that the increased expression
of a3b1 integrin after PMA treatment was accompanied by
increased exposure of the integrin on the melanocyte cell
surface (not shown).
Integrins in normal human melanocytes
183
Fig. 1. Immunoelectron microscopy was performed on dispase-separated epidermal sheets obtained from normal human skin as described
in Materials and Methods. Sheets were labeled with anti-b1 (a), -a3 (b), -av (c) and -a6 (d) mAbs and the appropriate gold conjugate. In
(a) the electron micrograph shows a basally located melanocyte sending dendrites among neighboring keratinocytes (d, dendrite). Inset: at
higher magnification, showing several gold particles on the melanocyte cell membrane (the number of gold particles observed on basal
keratinocytes with the antibody to b1 was higher than in melanocytes; this is not evident in (a) because of magnification problems). In (bd), gold particles (at arrows) appear scattered along the membrane of melanocytes (m, melanosomes). Gold particles (at arrowheads) are
visible on the cell membrane of neighboring keratinocytes (t, tonofilaments). Bar, 0.5 mm.
The PMA-dependent increase in a3 expression, was confirmed by northern blot analysis. Samples (20 mg) of total
RNA extracted from two different strains of melanocytes
cultured for 48 h in the presence of PMA, were hybridized
with an a3-specific human cDNA probe as described in
Materials and Methods. As shown in Fig. 5, the 5 kb a3
transcript was strongly increased in cells exposed to PMA
(lanes 2 and 4) compared to the untreated controls (lanes 1
and 3, respectively).
The surface exposure of both b1 and b3 integrins was
also studied by immunofluorescence on melanocytes cultured in the presence and in the absence of PMA. As previously described, melanocytes grown without PMA dis-
play a cobblestone appearence with short dendritic
processes, while, after PMA addition, cells acquire an elaborate dendritic morphology (De Luca et al., 1988b, 1993).
As shown in Fig. 6, in the absence of PMA (a-d), b1 integrins (d) displayed a rather uniform granular distribution
on the melanocyte cell surface; b1 integrins were occasionally aligned in tiny clusters along stress fibers with very
little evidence of their real association with genuine focal
contacts (d, at small arrows). Conversely, the avb3 integrin
was found exclusively at sites coincident with the termini
of F-actin microfilament bundles (a-b), in minute focal contacts (e.g. at arrows). This was confirmed by interference
reflection microscopy (IRM) and by co-staining with anti-
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G. Zambruno and others
Fig. 2. Immunoprecipitation.
Detergent lysates of metabolically
radiolabeled human melanocytes
(strain MP-31) grown in the
absence of PMA (−PMA) or
exposed for 48 h to 50 ng/ml of
PMA (+PMA) were
immunoprecipitated with antisera
and mAbs to different b integrin
subunits as described in Materials
and Methods. The eluates were
analysed by SDS-PAGE under
non-reducing (NR) and reducing
(R) conditions. Note the slight
increase in the intensity of the
bands of the a(s) subunit region
(at arrows) in melanocytes
exposed to PMA. Identical results
were obtained with four different
melanocyte strains. Protein-bound
radioactivity in cell lysates was
counted and equivalent amounts
of radioactivity were
immunoprecipitated. Molecular
mass standards are given in kDa
at the left and right.
Fig. 3. Immunoprecipitation.
Detergent lysates of metabolically
radiolabeled human melanocytes
(strain MP-31) grown in the
absence of PMA (−PMA) or
exposed for 4 8 h to 50 ng/ml of
PMA (+PMA) were
immunoprecipitated with mAbs to
different a subunits as described
in Materials and Methods. The
eluates were analysed by SDSPAGE under non-reducing (NR)
and reducing (R) conditions. The
different subunits and
heterodimers immunoprecipitated
are indicated in the figure. Note
the selective and strong increase,
both in NR and R conditions, of
the a3b1 integrin in cells
pretreated with PMA. Proteinbound radioactivity in cell lysates
was counted and equivalent
amounts of radioactivity were
immunoprecipitated. Molecular
mass standards are given in kDa
at the left and right.
bodies to talin and vinculin (not shown). In melanocytes
exposed to PMA (Fig. 6e-h), b1 and b3 integrins were found
also along and at the tip of dendritic processes (f and h,
respectively) and were present in tiny spots at the endings
of slender microfilament bundles (at arrows). Again, only
the avb3 complex seemed to be co-clustered with vinculin
Integrins in normal human melanocytes
Fig. 4. Immunoprecipitation.
Detergent lysates of surface radioiodinated human melanocytes (strain
MP-31) were immunoprecipitated
with mAbs to different α subunits as
described in Materials and Methods.
The eluates were analysed by SDSPAGE under non-reducing (NR)
conditions. The different subunits and
heterodimers immunoprecipitated are
indicated in the figure.
and talin, and thus associated with focal contacts (f, at
arrow).
Adhesion, spreading and migration assays
On the basis of integrin expression and topography, adhesion assays were performed on different substrata as
described in Materials and Methods. In preliminary experiments we noticed that: (i) melanocyte adhesion was
attained on different substrata after just 30 min, except on
laminin, which required at least 2 h; (ii) melanocytes
adhered better on fibronectin and vitronectin as compared
to laminin; (iii) the dendritic arborization was much more
elaborate on laminin as compared to fibronectin and vitronectin (see also Fig. 9); (iv) PMA pretreatment did not
change significantly the melanocyte adhesion on the dif-
185
Fig. 5. Northern blot
analysis. Samples (20
mg) of total RNA
extracted from two
different strain (MP30, lanes 1 and 2; MP27, lanes 3 and 4) of melanocytes cultured in the absence of PMA
(lanes 1 and 3) or exposed for 48 h to 50 ng/ml of PMA (lanes 2
and 4) were hybridized with the specific a3 cDNA probe as
described in Materials and Methods. Comparable amounts of
RNA were loaded in each lane as assessed by ethidium bromide
staining and hybridization with a specific cyclophilin cDNA probe
(not shown).
ferent substrata. As shown in Fig. 7A, the goat antiserum
to b1 integrins inhibited melanocyte adhesion to all substrata in a dose-dependent fashion. Interestingly, the mAb
to avb3 did not inhibit melanocyte adhesion, even when
cells were plated on vitronectin (Fig. 7B), whereas it inhibited the adhesion of human endothelial cells to vitronectin
(not shown). The different roles of b1 integrins were tested
by inhibition of adhesion by mAbs to the a subunits. As
shown in Fig. 8, melanocyte adhesion to fibronectin was
selectively inhibited by mAbs to the a5 subunit (A), while
adhesion to laminin was selectively inhibited by mAbs to
the a6 subunit (B). Monoclonal Abs to av, a3, a5 and a6
were not able to inhibit melanocyte adhesion to vitronectin
(Fig. 8C). These experiments were done in triplicate and
repeated at least twice with five different melanocyte
strains. Goat non-immune serum, as well as unrelated mon-
Fig. 6. Immunofluorescence localization of b3 (b,f) and b1 (d,h) in cultured human melanocytes without (a-d) and after (e-h) PMA
treatment. The corresponding localization of F-actin (stained by F-PHD, a,c,e and g) shows that b3 (b) is mostly found at the termini of
microfilament bundles (compare the position of arrows in a and b) while b1 is mostly diffuse and forms occasional clusters (d,h e.g. small
arrow). Integrin immunoreactivity is also found at the tips of dendrites in PMA-treated melanocytes (e-h). Bar, 10 mm.
186
G. Zambruno and others
A
A
β1 (antibody dilution)
B
B
αvβ3 (antibody dilution)
C
Fig. 7. Adhesion assay. Melanocytes (strain MP-30) were plated
on fibronectin (black bars), laminin (hatched bars) and vitronectin
(grey bars) in the presence of goat anti-serum to b1(A) and mAb
(LM 609) to the avb3 integrin (B) as described in Materials and
Methods. After 30 min (fibronectin and vitronectin) or 120 min
(laminin) at 37°C, cells were fixed and stained as described in
Materials and Methods. Each point was averaged from triplicates.
These experiments were repeated at least twice with five different
melanocyte strains.
oclonal antibodies, was ineffective in inhibiting melanocyte
adhesion.
We then tested the effects of different mAbs on
melanocyte spreading, as described in Materials and Methods. As shown in Fig. 9, melanocyte spread nicely both on
fibronectin (a) and on vitronectin (b). Note that cells plated
on laminin spread and sprouted short, but clearly evident,
dendritic processes (c). In marked contrast with their lack
of effect on melanocyte adhesion, mAbs to avb3 potently
inhibited melanocyte spreading both on fibronectin and vitronectin (d and e, respectively). Identical results were
obtained with mAbs to av (not shown). However, these Abs
were ineffective on melanocyte spreading and dendrite formation when cells were plated on laminin (f). Unrelated
mAbs were ineffective. Experiments were repeated twice
with three different melanocyte strains.
The migratory properties of normal human melanocytes
were assayed in Boyden chamber experiments. As shown
in Fig. 10, melanocytes migrated more to fibronectin(middle panel) and laminin- (lower panel) containing compartments than to vitronectin (upper panel). Melanocyte
migration to vitronectin was completely and selectively
blocked by mAbs to both av and avb3 (upper panel). A significant inhibition of melanocyte migration to fibronectin
Fig. 8. Adhesion assay. Melanocytes (strain MP-29) were plated
on fibronectin (A), laminin (B) and vitronectin (C) in the presence
of mAbs to a3 (P1B5), a5 (BIIG2), a6 (GoH3) and av (13C2), as
described in Materials and Methods. After 30 min (fibronectin and
vitronectin) or 120 min (laminin) at 37°C, cells were fixed and
stained as described in Materials and Methods. Each point was
averaged from triplicates. These experiments were repeated at
least twice with five different melanocyte strains.
and laminin was induced by mAbs to the a3 subunit (middle
and lower panels). Melanocyte migration to fibronectin and
laminin was also specifically inhibited by mAbs to a5 and
a6, respectively (middle and lower panels). In good agreement with the spreading inhibition data (Fig. 9), mAbs to
both av and avb3 gave good inhibition of melanocyte
migration to fibronectin but not to laminin (middle and
lower panels). Melanocytes did not migrate to control
medium containing bovine serum albumin. These experiments were done in triplicate and repeated at least twice
with three different melanocyte strains. Unrelated antibodies were ineffective.
Adhesion, spreading and migration assays were also performed with cells grown for 48 h in the presence of PMA.
Integrins in normal human melanocytes
187
Fig. 9. Spreading. Melanocytes (strain MP-31) were plated on fibronectin (a and d), on vitronectin (b and e) and on laminin (c and f) in
the absence (a-c) or in the presence (d-f) of the mAb to a vb3 (LM 609). Cells were fixed after 2 h and photographed as described in
Materials and Methods. Identical results were obtained with the mAb (13C2) to the av subunit. Experiments we repeated twice with three
different melanocyte strains. Bars, 25 mm.
No differences were noted in the effectiveness of the antibodies in the three assay types, compared to control cells.
DISCUSSION
The aim of this paper was to investigate the expression and
function of integrin receptors in normal human
melanocytes, both in vivo and in vitro. A large body of
information has been obtained on melanoma cells (see
Kramer et al., 1991a; Cheresh, 1991; Hart et al., 1991 for
recent reviews) but, surprisingly, very little data are available on their normal untransformed counterparts.
As previously reported (Albelda et al., 1990; Kramer et
al., 1991a; Zambruno et al., 1991), in vivo melanocytes
express members of the b1 family. The b1 subunit is presumably associated with a3 and a6, to form heterodimers
both endowed with laminin binding proper ties. Normal
human melanocytes isolated from reconstituted human epidermis and cultured in vitro, do indeed express the a3b1
and a6b1 heterodimers on their cell surface. In addition,
cultured melanocytes express a5b1 (the prototype
fibronectin receptor) and avb3 (the prototype vitronectin
receptor binding also to fibronectin, von Willebrand factor,
fibrinogen, thrombospondin and probably laminin; for
review see Ruoslahti, 1991). A novel receptor formed by
av and b1 subunits has recently been described (Vogel et
al., 1990; Bodary and McLean, 1990). In both metabolically and surface-radiolabeled melanocytes, av was found
to dimerize only with the b3 subunit. This behavior parallels the a6 capacity to associate with b1 only when the b4
subunit is not expressed (De Luca et al., 1990b; Pellegrini
et al., 1992). However, since the b3 subunit is not detectably
present in vivo, we cannot exclude the possibility of an
avb1 association in melanocytes in intact epidermis in situ.
It is worth considering that a broad integrin set is
expressed by malignant melanoma cells. The set includes,
in addition to integrins present in normal melanocytes in
situ, the following heterodimers: a1b1, a2b1, a4b1, a5b1,
a7b1 and avb3 (see Kramer et al., 1991a). This is not surprising, since metastatic melanoma cells travel across the
underlying dermis, gain access to lymphatic and blood vessels and form metastases. Indeed, the expression of a2b1
has been correlated to the capacity of melanoma cells to
reorganize collagen I fibrils (Klein et al., 1991); a4b1, able
to mediate lymphocyte adhesion to activated endothelial
cells (Elices et al., 1990), has been also suggested to mediate melanoma cell adhesion to vascular endothelia (Kramer
et al., 1991a); a1b1 (Kramer and Marks, 1989) and a7b1
(Kramer et al., 1991a,b) are additional receptors of basal
lamina components, and can be important in adhesion to
the microvascular subendothelium during hematogeneous
colonization; avb3, a vitronectin receptor that may also bind
laminin in microvascular endothelial cells (Kramer et al.,
1990), has been correlated with the invasive capacity of
metastatic melanoma cells (Albelda et al., 1990; Cheresh,
1991; Felding-Habermann et al., 1992; Seftor et al., 1992).
However, it is interesting to note that normal cultured
human melanocytes express a selective subset of melanoma
associated integrins, namely a5b1 and avb3. Similar behavior has been described for a5b1 and avb5 in proliferating
keratinocytes during wound healing (Adams and Watt,
1990; Marchisio et al., 1991; Pellegrini et al., 1992; De
Luca et al., 1992). Thus, it is tempting to speculate that the
expression of the latter two integrins might be correlated
not only to the migratory behavior of malignant cells, but
188
G. Zambruno and others
50
A
40
30
20
10
0
150
VN
+α5
+αvβ3
+αv
B
100
50
0
150
FN
+α 3
+α5
+α6
+αv
+α vβ 3
C
100
50
0
LN
+α3
+α5
+α6
+αv
+αvβ3
Fig. 10. Migration assay. Cell migration assays were
carried out in Boyden chambers as described in Materials and
Methods. The lower compartment was filled with media
containing vitronectin (A), fibronectin (B) and laminin (C).
Melanocytes (3×105 cells/ml) were added to the upper
compartment of the Boyden chamber in the absence (VN, FN and
LN) or in the presence of mAbs to a3 (P1B5), a5 (BIIG2), a6
(GoH3), av (13C2) and avb3 (LM 609). After 12 h cells were
fixed in ethanol, stained with toluidine blue, and 10 random fields
(u.f.) per filter were counted at ×160 with a microscope. Each
assay was carried out in triplicate and repeated at least three times.
also to the need for normal human melanocytes to adhere,
spread and migrate on the provisional matrix present in skin
wounds. This would allow melanocytes to follow migrating keratinocytes during wound healing, thus permitting
keratinocyte-dependent regulation of melanocyte function
to occur (see De Luca et al., 1993).
These putative roles of integrins expressed by cultured
melanocytes are further suggested by functional data.
Indeed, we have shown that the adhesion of melanocytes
to extracellular matrix proteins is mediated by specific integrins of the b1 family (a6b1 functioning as a melanocyte
laminin receptor and a5b1 as a fibronectin receptor), while
the avb3 complex is ineffective even on vitronectin. How-
ever, avb3 acquires a major role in regulating melanocyte
spreading onto substrata other than laminin. In addition,
while avb3 regulates normal melanocyte migration onto all
substrata but laminin, it acts in concert with specific b1
receptors in modulating such migratory properties. These
data are in good agreement with previous observations
showing that b1 and b3 integrins have different roles in the
adhesion and migration of vascular smooth muscle cells
(Clyman et al., 1992), and that collaborative interactions
among integrins can exist (Dejana et al., 1988; Charo et al.,
1990; Kramer et al., 1990; Bauer et al., 1992).
In summary, in normal human melanocytes: (i) adhesion
may involve only b1 integrins; (ii) spreading is mainly modulated by avb3; (iii) the b1 and b3 heterodimers cooperate
in regulating migration.
In our cells, and in contrast to data reported for melanoma
cells (see Kramer et al., 1991a), the avb3 complex does not
seem to interact with laminin. However, it is also well
known that, depending on the tissue, different isoforms of
laminin or laminin-related molecules do exist in basement
membranes (Hessle et al., 1984; Verrando et al., 1988;
Sanes et al., 1990; Yurchenko and Schittny, 1990; Carter
et al., 1991; Rousselle et al., 1991). Thus, the laminin binding properties of the avb3 integrin might be a peculiar feature of transformed melanocytes and, consequently, might
be important in the metastatic spreading of melanoma cells.
In this respect, it will be interesting to investigate the role
of avb3 in modulating the adhesive and migratory properties of normal melanocytes onto fibrinogen and von Willebrand factor.
We would like to add two additional observations. First,
since (i) antisera to b1 integrins are powerful inhibitors of
melanocyte adhesion to vitronectin; (ii) mAbs to all a subunits expressed by normal human melanocytes, including
av, are ineffective in inhibiting melanocytes adhesion to vitronectin; (iii) av does not associate with b1 in these cells;
(iv) there is a perfect parallelism between the effect of antiav and anti-avb3 mAbs, it is conceivable to postulate the
existence of a hitherto unidentified a subunit, associated
with b1, conferring vitronectin binding properties on these
cells. An alternative explanation could be that there is an
unknown regulatory mechanism conferring vitronectin
binding ability on a known b1 heterodimer expressed by
melanocytes.
Second, the a3b1 integrin seems to function, in cooperation with avb3, only in melanocyte migration. This effect
could be very important in regulating melanocyte migration
during embryonic development and in the recognition of
new basement membrane during wound healing. The a3b1
complex is strongly upregulated by PMA treatment, when
the sprouting of keratinocyte-contacting dendrites is maximal. Since a3b1 integrin has been recently demonstrated to
mediate cell-cell contact in human epidermis (De Luca et
al., 1990b; Larjava et al., 1990; Marchisio et al., 1991), it
will be interesting to investigate whether a3b1 does in fact
cooperate in mediating the intimate adhesion of
melanocytes and their dendrites with the surrounding keratinocytes. Moreover, it has been shown that cytokines can
modulate integrin expression on target cells (Santala and
Heino, 1991), and normal human keratinocytes in vivo and
in vitro are a source of several growth factors that are able
Integrins in normal human melanocytes
to regulate growth, migration and differentiation of surrounding melanocytes in a paracrine fashion and to modulate integrin expression on target cells (for recent reviews
see Luger and Schwarz, 1990; Santala and Heino, 1991; De
Luca et al., 1993). Among these factors, keratinocytes synthesize and secrete nerve growth factor (Di Marco et al.,
1991), which has been shown to regulate integrin
expression in PC12 cells (Rossino et al., 1990) and to modulate melanocyte migration and dendriticity (Yaar et al.,
1991; De Luca et al., 1993). In addition, keratinocytes synthesize a novel factor endowed with PMA-like activity,
which is also able to induce dendritic arborization in adjacent melanocytes (De Luca et al., 1993). Thus, it is tempting to speculate that during wound healing keratinocytes
might induce, through the secretion of this PMA-like
activity, dendrite formation and the overexpression of the
a3b1 heterodimer on surrounding melanocytes, rendering
these cells more able to migrate on the newly formed basement membrane and/or to establish contacts with basal keratinocytes.
This work was supported by Progetto Finalizzato ‘Biotecnologie e Biostrumentazione’ and ‘Applicazioni Cliniche della Ricerca
Oncologica’, Consiglio Nazionale delle Ricerche (CNR, Rome),
by Associazione Italiana per la Ricerca sul Cancro (AIRC,
Milano), by Ministero per l’Università e la Ricerca Scientifica e
Tecnologica (MURST, Roma) and by a grant from Regione Emilia
Romagna.
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(Received 3 December 1992 - Accepted 11 February 1993)