Modulation of vascular endothelial growthfactor receptors in melanocytes

Kim EJ, Park H-Y, Yaar M, Gilchrest BA. Modulation of vascularendothelial growth factor receptors in melanocytes.Exp Dermatol 2005: 14: 625–633. # Blackwell Munksgaard, 2005

Abstract: Vascular endothelial growth factor (VEGF) is constitutivelyproduced by keratinocytes, but has no known epidermal target cell. Wenow report that normal human melanocytes (Mc) maintained in serum-free, hormone-, and growth factor-supplemented medium lackingphorbol ester and choleragen constitutively express VEGF receptor-1(VEGFR-1), VEGFR-2, and neuropilin-1. Furthermore, stimulation ofMc with VEGF165 isoform leads to phosphorylation of VEGFR-2, thereceptor responsible for most of the VEGF-mediated effects inendothelial cells, suggesting that the receptor is functional. Interestingly,in Mc, VEGFR-2 expression is induced by ultraviolet irradiation andis downregulated by VEGF and tumor necrosis factor-a. Prolongedculture (>8 weeks) in the presence of phorbol ester abrogates VEGFR-2expression, explaining previous reports that Mc do not express VEGFR-1and VEGFR-2. These data suggest that VEGF may play a role inMc behavior in skin.

Ellen J. Kim, Hee-Young Park,Mina Yaar and Barbara A.Gilchrest

Department of Dermatology, Boston UniversitySchool of Medicine, Boston, MA, USA

Key words: melanocytes – phosphorylation – UV– vascular endothelial growth factor

Barbara A. Gilchrest, MDDepartment of DermatologyBoston University School of Medicine609 Albany Street, BostonMA 02118, USATel.: þ1 617 638 5538Fax: þ1 617 638 5550e-mail: bgilchre@bu.edu

Accepted for publication 27 April 2005

Introduction

Originally identified as an essential factor in bothnormal and aberrant angiogenesis, vascularendothelial growth factor (VEGF) is expressedby many cell types in tissue stroma (1). There arefive spliced variant isoforms of VEGF (alsoknown as VEGF-A), VEGF121, VEGF145,VEGF165, VEGF189, and VEGF206; and all exceptVEGF121 have a binding domain that interactswith cell-surface-associated heparan sulfate pro-teoglycans (2,3). A wide variety of tumors alsoexpress VEGF, which mediates tumor angio-genesis and lymphangiogenesis (4–7). In additionto the above isoforms, other VEGF familymembers exist, including placenta growth factor(PlGF), VEGF-B, VEGF-C, VEGF-D, andVEGF-E (8–11). Although cells can express severalisoforms simultaneously, the most abundant form isVEGF165, which binds several identified receptors,including the transmembrane tyrosine kinasereceptors VEGF receptor-1 (VEGFR-1, Flt-1),VEGF receptor-2 (VEGFR-2, flk-1/KDR), andthe cell-surface non-tyrosine kinase receptorneuropilin-1 (NP-1) (1–3,8).In endothelial cells (EC), both VEGFR-1 and

VEGFR-2 play a crucial role in normal blood

vessel development during embryogenesis(12,13). Binding of VEGF to VEGFR-2 resultsin dimerization and activation of the receptorthrough auto-phosphorylation of tyrosine resi-dues, and subsequent recruitment of signaltransduction pathway molecules including phos-pholipase C-gamma/protein kinase C, Nck,members of the Ras pathway, phosphatidylinositol 3-kinase, and nitric oxide synthase, result-ing in EC proliferation and migration (14–17).VEGFR-1 is also a high-affinity receptor forVEGF, but it demonstrates much weakerVEGF-dependent tyrosine phosphorylation thanVEGFR-2 (18). In ECs that express only VEGFR-1,VEGF fails to induceDNAsynthesis ormigration,in contrast to cells that express only VEGFR-2 (14).Recently, it has been shown that VEGFR-1 signalinginhibits VEGFR-2-mediated proliferation but notmigration (19–21).Neuropilin-1 was originally characterized as a

neuronal receptor for members of the collapsin/semaphorin family that have an inhibitory effecton neuronal guidance (22,23). Recently, it hasbeen shown to be an isoform-specific receptorfor VEGF165, and it was found to be expressedby cells such as ECs that are not of neuronal

Experimental Dermatology 2005: 14: 625–633 Copyright # Blackwell Munksgaard 2005Blackwell Munksgaard . Printed in Singapore EXPERIMENTAL DERMATOLOGY

ISSN 0906–6705

625

origin (24). NP-1 plays an important role on ECs,as a coreceptor by forming complexes withVEGFR-2 and enhancing VEGF binding(25,26). If VEGF binds to NP-1 in the absenceof VEGFR-2, no biological responses occur, sug-gesting that expression of NP-1 alone is insuffi-cient to mediate VEGF effects (3).VEGF is constitutively produced by keratino-

cytes (Kc) in the skin. Its production is up-regulated in psoriasis, wound healing, and otherstates of increased skin angiogenesis as well as byultraviolet (UV) irradiation (27–34). Kc-derivedVEGF affects dermal blood vessels (35), but todate it is not known whether VEGF affects thebehavior of epidermal cells. However, as manyother tumors, malignant melanomas, tumorsderived from epidermal melanocytes (Mc), wereshown to produce VEGF and express VEGFR-1,VEGFR-2, and NP-1 (4,36–38). Furthermore,Mc are neural crest-derived cells that produceand distribute pigment within the epidermis(39), and it has been recently shown that otherneuronal cells express VEGFRs and that VEGFstimulates axonal outgrowth, promotes neuronalcell survival, and plays a role in neural retinaldevelopment (40–43).Melanocytes reside in the basal layer of the

epidermis and each Mc is in contact withapproximately 36 surrounding Kc in a groupingtermed the ‘epidermal melanin unit’ (44) ManyKc-derived signaling molecules and cytokines,for which Mc express receptors, affect Mc survi-val, migration, and melanogenesis (45). Theseinclude basic fibroblast growth factor (46),alpha-MSH (47), endothelin-1 (48), nerve growthfactor (49), and tumor necrosis factor-a (TNF-a)(38), many of which also regulate neuronal cells.The expression of these factors is frequentlymodulated by stimuli of physiologic relevance tothe skin, such as UV irradiation (46–48,50).Keratinocyte expression of VEGF is also regu-

lated by various factors such as transforminggrowth factor-a (28), retinoids (51), hypoxia(52), and UV irradiation (33). UV irradiationis known to upregulate VEGF production inKc-derived cell lines, both directly through trans-cription factor activation and indirectly throughcytokine release (33,53). UV irradiation alsoupregulates Kc expression of TNF-a by a post-transcriptional mechanism (54). TNF-a mediateslymphocyte activation, apoptosis, and inflamma-tion. In ECs, TNF-a has been shown to bothinduce and decrease VEGFR-2 and NP-1 expres-sion, depending on the experimental conditionsand the type of cell studied (55,56), and to inhibitVEGFR-2 activation (57).

Because of the physical proximity of Mc to Kc,and because of the embryologic origin of Mc, weasked whether Mc were a potential target for Kc-derived VEGF, despite earlier reports suggestingthat this is not the case (58,59). We now reportthat early-passage normal human Mc maintainedin serum-free, hormone-, and growth factor-supplemented medium lacking phorbol ester andcholeragen constitutively expresses VEGFR-1,VEGFR-2, and NP-1. Furthermore, we foundthat UV irradiation induces VEGFR-2 in Mc,while TNF-a and VEGF downregulate the levelof this receptor. Finally, the phorbol ester,12-O-tetradecanoylphorbol-13-acetate (TPA),frequently used as a non-physiologic but growth-promoting supplement in Mc cultures (58–60)initially induces VEGFR-2 level, but prolongedculturing abrogatesVEGFexpression. These studiessuggest that VEGF may play as yet unrecognizedroles in Mc development and function innormal skin.

Materials and methods

Melanocyte culture

Human neonatal foreskins Mc were cultured as described pre-viously (49,53). Primary cultures of Mc were subcultured andmaintained in calcium-free Medium 199 (Gibco BRL,Gaithersburg, MD, USA) supplemented with 2% fetal bovineserum, 10mg/ml insulin, 10�9M triiodothyronine, 10mg/ml trans-ferrin, 10ng/ml EGF, and 55mM inositol for at least 24h beforeany studies. Bovine pituitary extract and basic fibroblast growthfactor were not used in the Mc subcultures. To determine theeffect of phorbol esters on VEGFR-2 expression, Mc weretreated for various periods of time with either diluent (DMSO)alone or 50ng/ml (81 nM) TPA (Sigma-Aldrich, St. Louis, MO,USA). Human umbilical vein ECs (HUVEC) (BioWhittaker,Walkersville, MD, USA) and human melanoma lines MC(derived originally from primary recurrent melanoma) (61) andRU (derived originally from metastatic melanoma) (61) (a kindgift of H.R. Byers, Boston, MA, USA) were used as positivecontrols. All experiments were reproduced at least two times.

Reagents

Mouse monoclonal antibodies against VEGFR-2 (sc-6251,recognizes amino acids 1158–1345) and NP-1 (A-12, recognizesamino acids 570–855) were obtained from Santa CruzBiotechnology (Santa Cruz, CA, USA). VEGFR-1 mousemonoclonal antibody (Flt-11, recognizes amino acids 1–251)was obtained from Sigma-Aldrich. Phospho-tyrosine mousemonoclonal antibody (P-Tyr-100) was obtained from CellSignaling Technology (Beverly, MA, USA). Recombinanthuman VEGF165 was obtained from R&D Systems(Minneapolis, MN, USA). Recombinant TNF-a was purchasedfrom Sigma-Aldrich.

UV irradiation studies

A solar simulator (Spectral Energy Corporation, Westwood,NJ, USA) housing an appropriately filtered 1 kW xenon arc

Kim et al.

626

lamp (XMN 1000-21; Optical Radiation, Azusa, CA, USA)adjusted to 2� 10�4W cm�2 was used to irradiate cells inphosphate-buffered saline (PBS) through the Petri dish plasticcover. This system delivers a spectral output virtually identicalto that of terrestrial sunlight (54). Cells received a physio-logic dose of 30mJ/cm2 as metered at 285� 5 nm with aresearch radiometer (model IL1700A; International Light,Newburyport, MA, USA) fitted with a UVB probe (detectorSSE 240, diffuser W, filter UVB). Sham-irradiated cells werehandled identically but were placed under aluminum foil coverduring the irradiation. Cells were harvested 48 and 120 h afterirradiation.

Western blot analysis

Cells were extracted in harvest buffer (�1 PBS, 1% Triton X,1mg/ml aprotinin, 100 mM okadaic acid, 1mM PMSF), andprotein concentration was determined by the Bradford proteinassay. Protein (100mg/ml) was boiled at 100�C in sodium dodecylsulfate (SDS) sample buffer for 2min, electrophoresed on 7.5%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels, andtransferred to polyvinyldifluoridine membranes. These wereincubated for 1 h at 37�C and then overnight at 4�C with theappropriate primary mouse monoclonal antibody. Membraneswere washed with �1 PBS/0.1% Tween-20 (PBS/Tween), andincubated with horseradish peroxidase-conjugated antimousesecondary antibody (Amersham Pharmacia Biotechnology,Buckinghamshire, UK; 1 : 2000) at room temperature for 1 hand washed with PBS/Tween. Peroxidase activity was visualizedwith a chemiluminescence substrate system (ECLþ, AmershamPharmacia Biotechnology) and exposed to X-OMAT film(Kodak, Rochester, NY, USA).

Receptor-activation studies

Subconfluent Mc were incubated overnight in serum-freeDulbecco’s Modified Eagle (DME) medium. Cells were thenstimulated with either diluent alone or VEGF165 (20 ng/ml) for5min at 37�C. Cells were washed with ice-cold �1 PBS andwere extracted with harvest buffer as described above with theaddition of 1mM sodium orthovanadate and 50mM sodiumfluoride. Lysates were subjected to SDS-PAGE, and immuno-blots were reacted in parallel with antibodies for VEGFR-2 andantiphosphotyrosine.

Results

Newborn human Mc express VEGFR-1, VEGFR-2,and NP-1

To determine whether normal human Mc expressVEGFRs, second passage Mc were grown instandard medium without phorbol esters orcholeragen until near-confluent and processed forwestern immunoblot analysis with the appropri-ate monoclonal antibodies for VEGFR-2(sc-6251, Fig. 1a), VEGFR-1 (Flt-11, Fig. 1b),and NP-1 (NP-1, Fig. 1c). Immunoblotting withthe VEGFR-2 antibody demonstrated that Mcexpress the same 200 kDa band as seen in thepositive-control HUVEC and melanoma cellsMC and RU (Fig. 1a). The HUVEC also express

an additional band at 220 kDa, which hasbeen shown to correspond to a more extensivelyN-linked glycosylated form (62). Similarly,immunoblot analysis for VEGFR-1 demon-strated that Mc express the same 160 kDa bandas seen in the HUVEC and melanoma cells(Fig. 1b). An additional 180 kDa band corres-ponding to a more extensively glycosylated formof VEGFR-1 was detected in HUVEC (Fig. 1b)(62). Kc did not show a detectable level ofVEGFR-1 (data not shown).NP-1 is not a tyrosine–kinase receptor, nor

does it have intrinsic signaling activity whenexposed to VEGF (3). However, when coex-pressed with VEGFR-2, it acts as a coreceptorfor VEGF (24). Melanocytes express the 140 kDaprotein, corresponding to the known molecularmass of NP-1 (Fig. 1c). A same-sized band isdemonstrated in the positive-control HUVECand melanoma lines. Thus, cultured newbornhuman early-passage Mc express at least threeof the known VEGFR proteins: VEGFR-2,VEGFR-1, and NP-1. Of note, receptor expres-sion level is at least as high in Mc as in thecontrol cell lines.

220 kDa

HUVEC Mc

VEGFR-2

VEGFR-1

MC RU

200 kDa

180 kDa

b

c

a

160 kDa

140 kDa

NP-1

Coomassieblue stain

Figure 1. Melanocytes (Mc) constitutively express a form ofvascular endothelial growth factor (VEGFR-2), VEGFR-1,and neuropilin-1 (NP-1). Near-confluent cultures of normalhuman Mc were maintained in serum-free, growth factor- andhormone-supplemented M199 medium without phorbol ester orcholeragen. Hundred micrograms of total cellular proteinswere harvested and processed for immunoblotting with (a)anti-VEGFR-2 (KDR) (b) anti-VEGFR-1 (Flt-1), or (c) anti-neuropilin-1 (NP-1) antibodies, respectively. Human umbilicalvein endothelial cell cultures (HUVEC) and melanoma cell linesMC and RU were harvested in parallel for positive controls(50 mg each).

Modulation of VEGF receptors

627

VEGF165 activates Mc VEGFR-2

In ECs, VEGFR-2 is the major receptor respon-sible for VEGF-mediated effects such as pro-liferation and migration. Because VEGFR-2 isa tyrosine–kinase receptor, we asked whetherVEGFR-2 on Mc is phosphorylated uponVEGF stimulation. Melanocytes were treatedwith diluent alone or VEGF (50 ng/ml) for5min. As before, Western blot analysis revealedthat both diluent alone and VEGF-treated Mcexpress the 200 kDa band representing the bisglycosylated form of VEGFR-2 (Fig. 2), asshown above. Interestingly, immunoblotting per-formed in parallel with an antiphosphotyrosineantibody revealed that when stimulated withVEGF, both HUVEC and Mc express a bandcorresponding to the 220 VEGFR-2, suggestingthat in Mc although the level of the fully glyco-sylated VEGFR-2 is below the level of detectionwhen using anti-VEGFR-2 antibody, neverthe-less, this receptor is present in Mc and becomesactivated by VEGF.

Prolonged treatment with phorbol esterssuppresses VEGFR-2 and NP-1 expression in Mc

Previous reports failed to detect VEGFR-2 innormal human Mc (58,59). However, the culturesystem used in these reports contained TPA, anadditive known to have major effects on geneexpression, and Mc were maintained throughmultiple (up to 60) passages (58,59). To deter-mine the effect of TPA on VEGFR-2 expression,Mc were treated with TPA (50 ng/ml¼ 81 nM) ordiluent alone for up to 9 weeks. Western blotanalysis showed that VEGFR-2 levels in Mc arenot modulated within hours of TPA stimulation(Fig. 3a), but are increased within 5 days and 1

month (Fig. 3b). However, after 8 or more weeksof cultivation in the presence of TPA, VEGFR-2cannot be detected (Fig. 3c).

UV irradiation upregulates VEGFR-2 expressionin melanocytes

To determine the effect of UV irradiation on McVEGFR-2 expression, cells were exposed to30mJ/cm2 of solar-simulated UV irradiation orsham irradiation and processed for Western blotanalysis. Within 48 h, VEGFR-2 level in UV-irradiated Mc was twice that in sham-irradiatedcontrols (Fig. 4). Within 120 h, VEGFR-2 expres-sion approached baseline levels.

HUVEC Mc

VEGFR-2 -VEGFR-2P PVEGFR-2 -VEGFR-2

Figure 2. Vascular endothelial growth factor (VEGF)activates VEGFR receptor-2 (VEGFR-2) on melanocytes (Mc).Subconfluent cultures of normal human Mc were harvested, andtotal cellular proteins were processed for immunoblot analysis withVEGFR-2 antibody and antiphosphotyrosine antibody in parallel.HUVEC cultures were processed in as a positive control.

TPA 0 0.5 h

5 days

TPA

b

c

a

VEGFR-2

Coomassieblue

Coomassieblue

TPA exposure

VEGFR-2

2 week 8 week 9 week

– –+ +

1 month

1.5 h 2.0 h1 h

VEGFR-2

Coomassieblue

Figure 3. Vascular endothelial growth factor receptor-2(VEGFR-2) modulations by 12-O-tetradecanoylphorbol-13-acetate (TPA). Normal human melanocytes (Mc) were treatedwith 50 ng/ml (81 nM) TPA or diluent for different times. Totalcellular proteins were harvested after exposure and processedfor immunoblot analysis with an anti-VEGFR-2 antibody. Noconsistent modulation of VEGFR-2 level was observed up to2 h after stimulation (a). However, VEGFR-2 levels were higherin cells treated for 5 days with TPA as compared to diluent-treated control, and even higher 1 month after TPA treatment(b). Mc maintained for up to 9 weeks in the presence of TPAlost VEGFR-2 expression within 8 weeks of culturing.

Kim et al.

628

VEGF165 downregulates Mc VEGFR-2 expression

To determine whether exposure to VEGF affectsMc VEGFR-2 levels, Mc were stimulated withVEGF165 (50 ng/ml) or diluent alone (control) for48 h. Western blot analysis revealed that VEGFdownregulated VEGFR-2 level in Mc to half itsbaseline level within 48 h, suggesting a possiblenegative feedback loop (Fig. 5).

TNF-� downregulates VEGFR-2 expression in Mc

TNF-a is a pro-inflammatory cytokine producedby a wide variety of cells, including Kc, after UV

irradiation (54). In various EC lines, TNF-a isreported to downregulate VEGFR-2 transcrip-tion and inhibit its phosphorylation (55,57), orconversely upregulate its expression (56), depend-ing on experimental conditions. To determineTNF-a effects, Mc were treated with 0, 10, and40 ng/ml TNF-a, doses routinely used to stimu-late cultured cells (63,64), and VEGFR-2 levelwas determined. Within 72 h of TNF-a exposure,Mc demonstrated a dose-dependent decrease inVEGFR-2 levels of more than 50% at the higherdose (Fig. 6).

Discussion

Keratinocyte-derived VEGF is important incutaneous angiogenesis during wound healingand tumorigenesis and in pathologic conditionslike psoriasis. However, it is unknown whetherany epidermal cell is a target for VEGF. Becausemany Kc-derived cytokines affect Mc, andbecause other neural crest-derived cells are regu-lated by VEGF, we speculated that Mc may be atarget for VEGF. To explore this possibility, wefirst examined VEGFR expression and modula-tion in cultured newborn human Mc.Our experiments demonstrate that Mc express

three VEGFRs at the protein level: the twotyrosine–kinase receptors VEGFR-2 and VEGFR-1,and NP-1, a VEGFR-2 coreceptor. This is incontrast to previous reports that after prolongedpassage, human Mc do not express VEGFR-1 orVEGFR-2 (58,59). However, these studiesmaintained Mc in the presence of the phorbolester, TPA, while we maintained human Mc in

VEGFR-2

UV

48 h 120 h

– + – +

Coomassieblue

Figure 4. UV irradiation upregulates vascular endothelialgrowth factor receptor-2 (VEGFR-2) expression. Normalhuman melanocytes (Mc) were exposed to 30mJ/cm2 solarsimulated UV irradiation or were sham irradiated. 48 and120 h postirradiation, total cellular proteins were harvestedand processed for immunoblot analysis with an anti-VEGFR-2 antibody. Within 48 h, VEGFR-2 in UV-irradiated Mc wasinduced 200% as compared to sham-irradiated Mc, as deter-mined by densitometry. Densitometry was performed on theautoradiogram presented in the figure. Within 120h, VEGFR-2in sham-irradiated Mc was comparable to that of UV-irradiatedcells. Representative results from at least two independentexperiments are presented.

VEGFR-2

Coomassieblue

VEGF – +

Figure 5. Vascular endothelial growth factor receptor-2(VEGFR-2) level stimulation within 120 h decreases melanocyte(Mc) VEGFR-2 expression. Normal human Mc were treatedwith VEGF (50 ng/ml) or diluent as control for 48 h. Totalcellular proteins were harvested and processed for immunoblotanalysis that was reacted with anti-VEGFR-2 antibody. Within48 h, VEGF decreased the level of VEGFR-2 by 50%, as deter-mined by densitometry measured on the autoradiogram pre-sented in the figure. Representative results from at least twoseparate experiments are shown.

TNF-α(ng/ml)

VEGFR-2

Coomassieblue

0 10 40

Figure 6. Tumor necrosis factor-a (TNF-a) stimulation down-regulates melanocyte (Mc) vascular endothelial growth factor-2(VEGFR-2) levels. Normal human Mc were stimulated withincreasing concentrations of TNF-a or diluent. Seventy-twohours after treatment, total cellular proteins were harvestedand processed for immunoblot analysis using an anti-VEGFR-2antibody. As determined by densitometry on autoradiogrampresented in the figure, TNF-a downregulated VEGFR-2 levelin a dose-dependent manner with maximum 75% downregula-tion observed at 40 ng/ml. Representative results from at leasttwo independent experiments are shown.

Modulation of VEGF receptors

629

serum-free, growth hormone-supplementedmedium lacking TPA and analyzed only early-passage cells (less than two postprimary popula-tion doublings). We selected this experimentalsystem because profound changes are known tooccur in cultured cells even after relatively fewpopulation doublings. For example, newbornhuman fibroblasts markedly downregulateepidermal growth factor receptor expressionafter only four population doublings (65).Additionally, it has been observed that duringprolonged cultivation in phorbol ester containingmedium, Mc lose E-cadherin expression (66).Thus, prolonged culture may substantially affectreceptor expression. TPA, which initially activatesand then depletes protein kinase C (67), greatlyfacilitates long-term Mc culture (60), and has alsobeen shown to profoundly affect Mc, inducing adendritic morphology and slender spindle shape tothe cells (68), altering growth factor responsiveness(69), and decreasing melanogenic capacity (68,70).At least on smooth muscle cells, TPA specificallydownregulates the expression of endothelin-1receptor (71), suggesting that prolonged TPAexposure may similarly affect the expressionof other cell-surface receptors, including theVEGFRs. Our experiments demonstrate that theinitially strong VEGFR-2 expression is lost after8 weeks of cultivation in the presence of TPA.Whether VEGFR-2 downregulation is due princi-pally to in vitro aging, to TPA or to the combin-ation, was not explicitly addressed in our studydesign, as prolonged serial cultivation in theabsence of TPA and/or artificially elevated levelsof cAMP is extremely difficult (71), and we main-tainedMc under basal conditions for only 1 monthafter generating sufficient cells for the TPAexperiments.Western blot analysis with anti-VEGFR-2

antibodies failed to detect the fully glycosylatedreceptor in Mc. Nevertheless, VEGF stimulationclearly induced phosphorylation of the fullyglycosylated VEGFR-2 isoform in Mc, asdetected by an antiphosphotyrosine antibody,indicating receptor activation. It is not clearwhy the fully glycosylated form of VEGFR-2was not detected in Mc, but it is likely thatthe level of this isoform is low and/or the affinityof anti-VEGFR-2 antibody for its epitope islower than that of the antiphosphotyrosineantibody. Regardless, our finding suggeststhat VEGFR-2 in Mc is activated by VEGFstimulation. Further experiments are required todetermine VEGF-induced signaling in Mc andhow VEGFR-2 activation modulates Mcfunction.

We show that VEGFR-2 expression on Mc ismodulated by several factors. UV irradiationhas been shown to induce VEGF synthesis inKc (32) and Kc-derived cell lines (53), and ourdata demonstrate that UV irradiation alsoupregulates VEGFR-2 expression in Mc. Incontrast, we show that VEGF and TNF-a,Kc-derived cytokines that are upregulated byUV irradiation (32,72), both downregulateVEGFR-2 levels. Thus, the overall effect of UVirradiation on VEGF signaling in Mc is likely tobe complex.

We have examined the possible role of VEGFon Mc function. Our preliminary resultssuggested that VEGF did not increase themelanin level, the major differentiated productof Mc (data not shown). Moreover, VEGFdid not alter Mc proliferation or dendricity.Time-lapse photographs did not show aneffect on Mc migration. Although these initialresults suggest that VEGF may not affect Mcproliferation or differentiation, it is importantto keep in mind that unlike Mc in vivo, culturedMc are supplemented with several growth factorsand hormones, and their effect many thus maskVEGF effects on these parameters. More experi-ments are required to delineate this point.

This report demonstrates that like other neuralcrest derived cells (40–43,73), normal human Mcexpress VEGFRs. The functional significance ofVEGFR expression in Mc and whether VEGFsignaling plays any role in malignant transforma-tion to melanoma remain to be determined.Nevertheless, our results suggest that VEGF ofepidermal origin may affect not only cutaneousangiogenesis, but may also exert paracrine effectson Mc.

References

1. Ferrara N. Vascular endothelial growth factor:Molecular and biological aspects. In: Cleasson-Welsh,L, ed. Vascular Growth Factors and Angiogenesis.Berlin: Springer-Verlag, 1999: 1–29.

2. Petrova T V, Makinen T, Alitalo K. Signaling via vas-cular endothelial growth factor receptors. Exp Cell Res1999: 253: 117–130.

3. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z.Vascular endothelial growth factor (VEGF) and itsreceptors. FASEB J 1999: 13: 9–22.

4. Claffey K P, Brown L F, del Aguila L F et al.Expression of vascular permeability factor/vascularendothelial growth factor by melanoma cells increasestumor growth, angiogenesis, and experimental metasta-sis. Cancer Res 1996: 56: 172–181.

5. Salven P, Heikkila P, Joensuu H. Enhanced expressionof vascular endothelial growth factor in metastatic mel-anoma. Br J Cancer 1997: 76: 930–934.

Kim et al.

630

6. Skobe M, Hawighorst T, Jackson D G et al. Inductionof tumor lymphangiogenesis by VEGF-C promotesbreast cancer metastasis. Nat Med 2001: 7: 192–198.

7. Skobe M, Hamberg L M, Hawighorst T et al.Concurrent induction of lymphangiogenesis, angiogen-esis, and macrophage recruitment by vascular endothe-lial growth factor-C in melanoma. Am J Pathol 2001:159: 893–903.

8. Carmeliet P, Moons L, Luttun A et al. Synergismbetween vascular endothelial growth factor and placen-tal growth factor contributes to angiogenesis andplasma extravasation in pathological conditions. NatMed 2001: 7: 575–583.

9. Scrofani S D, Fabri L J, Xu P, Maccarone P, Nash A D.Purification and refolding of vascular endothelialgrowth factor-B. Protein Sci 2000: 9: 2018–2025.

10. Yonekura H, Sakurai S, Liu X et al. Placenta growthfactor and vascular endothelial growth factor B and Cexpression in microvascular endothelial cells andpericytes. Implication in autocrine and paracrineregulation of angiogenesis. J Biol Chem 1999: 274:35172–35178.

11. Clauss M. Molecular biology of the VEGF and theVEGF receptor family. Semin Thromb Hemost 2000:26: 561–569.

12. Fong G H, Rossant J, Gertsenstein M, Breitman M L.Role of the Flt-1 receptor tyrosine kinase in regulatingthe assembly of vascular endothelium. Nature 1995:376: 66–70.

13. Shalaby F, Rossant J, Yamaguchi T P et al. Failure ofblood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995: 376: 62–66.

14. Shibuya M, Ito N, Claesson-Welsh L. Structure andfunction of vascular endothelial growth factor recep-tor-1 and -2. Curr Top Microbiol Immunol 1999: 237:59–83.

15. Guo D, Jia Q, Song H Y, Warren R S, Donner D B.Vascular endothelial cell growth factor promotes tyro-sine phosphorylation of mediators of signal transduc-tion that contain SH2 domains. Association withendothelial cell proliferation. J Biol Chem 1995: 270:6729–6733.

16. Shen B Q, Lee D Y, Zioncheck T F. Vascular endothe-lial growth factor governs endothelial nitric-oxidesynthase expression via a KDR/Flk-1 receptor and aprotein kinase C signaling pathway. J Biol Chem 1999:274: 33057–33063.

17. Dayanir V, Meyer R D, Lashkari K, Rahimi N.Identification of tyrosine residues in vascular endothe-lial growth factor receptor-2/FLK-1 involved in activa-tion of phosphatidylinositol 3-kinase and cellproliferation. J Biol Chem 2001: 276: 17686–17692.

18. Seetharam L, Gotoh N, Maru Y, Neufeld G,Yamaguchi S, Shibuya M. A unique signal transductionfrom FLT tyrosine kinase, a receptor for vascularendothelial growth factor VEGF. Oncogene 1995: 10:135–147.

19. Rahimi N, Dayanir V, Lashkari K. Receptor chimerasindicate that the vascular endothelial growth factorreceptor-1 (VEGFR-1) modulates mitogenic activity ofVEGFR-2 in endothelial cells. J Biol Chem 2000: 275:16986–16992.

20. Zeng H, Dvorak H F, Mukhopadhyay D. Vascularpermeability factor (VPF) /vascular endothelial growthfactor (VEGF) peceptor-1 down-modulates VPF/VEGFreceptor-2-mediated endothelial cell proliferation, butnot migration, through phosphatidylinositol 3-kinase-

dependent pathways. J Biol Chem 2001: 276:26969–26979.

21. Zeng H, Sanyal S, Mukhopadhyay D. Tyrosine residues951 and 1059 of vascular endothelial growth factorreceptor-2 (KDR) are essential for vascular permeabil-ity factor/vascular endothelial growth factor-inducedendothelium migration and proliferation, respectively.J Biol Chem 2001: 276: 32714–32719.

22. Kolodkin A L, Levengood D V, Rowe E G, Tai Y T,Giger R J, Ginty D D. Neuropilin is a semaphorin IIIreceptor. Cell 1997: 90: 753–762.

23. Song H, Ming G, He Z et al. Conversion of neuronalgrowth cone responses from repulsion to attraction bycyclic nucleotides. Science 1998: 281: 1515–1518.

24. Soker S, Takashima S, Miao H Q, Neufeld G,Klagsbrun M. Neuropilin-1 is expressed by endothelialand tumor cells as an isoform-specific receptor for vas-cular endothelial growth factor. Cell 1998: 92: 735–745.

25. Fuh G, Garcia K C, de Vos A M. The interaction ofneuropilin-1 with vascular endothelial growth factorand its receptor flt-1. J Biol Chem 2000: 275:26690–26695.

26. Whitaker G B, Limberg B J, Rosenbaum J S. Vascularendothelial growth factor receptor-2 and neuropilin-1form a receptor complex that is responsible for thedifferential signaling potency of VEGF (165) andVEGF (121). J Biol Chem 2001: 276: 25520–25531.

27. Brown L F, Yeo K T, Berse B et al. Expression ofvascular permeability factor (vascular endothelialgrowth factor) by epidermal keratinocytes duringwound healing. J Exp Med 1992: 176: 1375–1379.

28. Detmar M, Brown L F, Claffey K P et al.Overexpression of vascular permeability factor/vascularendothelial growth factor and its receptors in psoriasis.J Exp Med 1994: 180: 1141–1146.

29. Detmar M, Yeo K T, Nagy J A et al. Keratinocyte-derived vascular permeability factor (vascularendothelial growth factor) is a potent mitogen fordermal microvascular endothelial cells. J InvestDermatol 1995: 105: 44–50.

30. Lauer G, Sollberg S, Cole M et al. Expression andproteolysis of vascular endothelial growth factor isincreased in chronic wounds. J Invest Dermatol 2000:115: 12–18.

31. Gille J, Reisinger K, Asbe-Vollkopf A, Hardt-Weinelt K,Kaufmann R. Ultraviolet-A-induced transactivation ofthe vascular endothelial growth factor gene inHaCaT keratinocytes is conveyed by activator protein-2 transcription factor. J Invest Dermatol 2000: 115:30–36.

32. Brauchle M, Funk J O, Kind P, Werner S. Ultraviolet Band H2O2 are potent inducers of vascular endothelialgrowth factor expression in cultured keratinocytes. JBiol Chem 1996: 271: 21793–21797.

33. Mildner M, Weninger W, Trautinger F, Ban J,Tschachler E. UVA and UVB radiation differentiallyregulate vascular endothelial growth factor expressionin keratinocyte-derived cell lines and in human keratino-cytes. Photochem Photobiol 1999: 70: 674–679.

34. Weninger W, Uthman A, Pammer J et al. Vascularendothelial growth factor production in normal epider-mis and in benign and malignant epithelial skin tumors.Lab Invest 1996: 75: 647–657.

35. Supp D M, Supp A P, Bell S M, Boyce S T. Enhancedvascularization of cultured skin substitutes geneticallymodified to overexpress vascular endothelial growthfactor. J Invest Dermatol 2000: 114: 5–13.

Modulation of VEGF receptors

631

36. Liu B, Earl H M, Baban D et al. Melanoma cell linesexpress VEGF receptor KDR and respond to exogen-ously added VEGF. Biochem Biophys Res Commun1995: 217: 721–727.

37. Herold-Mende C, Steiner H H, Andl T et al. Expressionand functional significance of vascular endothelialgrowth factor receptors in human tumor cells. LabInvest 1999: 79: 1573–1582.

38. Straume O, Akslen L A. Expression of vascularendothelial growth factor, its receptors (FLT-1, KDR)and TSP-1 related to microvessel density and patientoutcome in vertical growth phase melanomas. Am JPathol 2001: 159: 223–235.

39. Jimbow K, Quevedo W C, Fitzpatrick T B, Szabo G.Some aspects of melanin biology: 1950–1975. J InvestDermatol 1976: 67: 72–89.

40. Sondell M, Sundler F, Kanje M. Vascular endothelialgrowth factor is a neurotrophic factor which stimulatesaxonal outgrowth through the flk-1 receptor. Eur JNeurosci 2000: 12: 4243–4254.

41. Sondell M, Kanje M. Postnatal expression of VEGFand its receptor flk-1 in peripheral ganglia.Neuroreport 2001: 12: 105–108.

42. Robinson G S, Ju M, Shih S C et al. Nonvascular rolefor VEGF. VEGFR-1, -2 activity is critical for neuralretinal development. FASEB J 2001: 15: 1215–1217.

43. Matsuzaki H, Tamatani M, Yamaguchi A et al.Vascular endothelial growth factor rescues hippocampalneurons from glutamate-induced toxicity: signal trans-duction cascades. FASEB J 2001: 15: 1218–1220.

44. Jimbow K, Prota G, Fitzpatrick T B. Biology of melano-cytes. In: Freedberg I M, Austen K F, Goldsmith L A,Katz S I, Fitzpatrick T B, eds. Dermatology in generalmedicine. New York: McGraw-Hill, 1999: 192–219.

45. Gilchrest B A, Eller M S, Yaar M. The photobiology ofthe tanning response. In: Nordlund J J, Hearing V J,King R A, Ortonne J P, eds. Pathophysiology. NewYork: Oxford University Press, 1998: 359–372.

46. Halaban R, Langdon R, Birchall N et al. Basic fibro-blast growth factor from human keratinocytes is anatural mitogen for melanocytes. J Cell Biol 1988: 107:1611–1619.

47. Schauer E, Trautinger F, Kock A et al.Proopiomelanocortin-derived peptides are synthesizedand released by human keratinocytes. J Clin Invest1994: 93: 2258–2262.

48. Imokawa G, Yada Y, Miyagishi M. Endothelinssecreted from human keratinocytes are intrinsic mito-gens for human melanocytes. J Biol Chem 1992: 267:24675–24680.

49. Yaar M, Grossman K, Eller M, Gilchrest B A. Evidencefor nerve growth factor-mediated paracrine effects inhuman epidermis. J Cell Biol 1991: 115: 821–828.

50. Tron V A, Coughlin M D, Jang D E, Stanisz J,Sauder D N. Expression and modulation of nervegrowth factor in murine keratinocytes (PAM 212).J Clin Invest 1990: 85: 1085–1089.

51. Lachgar S, Charveron M, Gall Y, Bonafe J L.Inhibitory effects of retinoids on vascular endothelialgrowth factor production by cultured humanskin keratinocytes. Dermatology 1999: 199 (Suppl. 1):25–27.

52. Detmar M, Brown L F, Berse B et al. Hypoxia regulatesthe expression of vascular permeability factor/vascularendothelial growth factor (VPF/VEGF) and itsreceptors in human skin. J Invest Dermatol 1997: 108:263–268.

53. Kosmadaki M G, Yaar M, Arble B L, Gilchrest B A.UV induces VEGF through a TNF-alpha independentpathway. FASEB J 2003: 17: 446–448.

54. LeverkusM, YaarM, EllerM S, Tang EH, Gilchrest B A.Post-transcriptional regulation of UV induced TNF-alphaexpression. J Invest Dermatol 1998: 110: 353–357.

55. Patterson C, Perrella M A, Endege W O, Yoshizumi M,Lee M E, Haber E. Downregulation of vascularendothelial growth factor receptors by tumor necrosisfactor-alpha in cultured human vascular endothelialcells. J Clin Invest 1996: 98: 490–496.

56. Giraudo E, Primo L, Audero E et al. Tumor necrosisfactor-alpha regulates expression of vascular endothelialgrowth factor receptor-2 and of its co-receptor neuropi-lin-1 in human vascular endothelial cells. J Biol Chem1998: 273: 22128–22135.

57. Guo D Q, Wu L W, Dunbar J D et al. Tumor necrosisfactor employs a protein-tyrosine phosphatase to inhibitactivation of KDR and vascular endothelial cell growthfactor-induced endothelial cell proliferation. J BiolChem 2000: 275: 11216–11221.

58. Gitay-Goren H, Halaban R, Neufeld G. Human mela-noma cells but not normal melanocytes express vascularendothelial growth factor receptors. Biochem BiophysRes Commun 1993: 190: 702–708.

59. Graeven U, Fiedler W, Karpinski S et al. Melanoma-associated expression of vascular endothelial growthfactor and its receptors FLT-1 and KDR. J CancerRes Clin Oncol 1999: 125: 621–629.

60. Eisinger M, Marko O. Selective proliferation of normalhuman melanocytes in vitro in the presence of phorbolester and cholera toxin. Proc Natl Acad Sci USA 1982:79: 2018–2022.

61. Byers HR, Etoh T, Doherty J R, Sober A J,MihmMC Jr.Cell migration and actin organization in culturedhuman primary, recurrent cutaneous and metastaticmelanoma. Time-lapse and image analysis. Am JPathol 1991: 139: 423–435.

62. Takahashi T, Shibuya M. The 230 kDa matureform of KDR/Flk-1 (VEGF receptor-2) activates thePLC-gamma pathway and partially induces mitoticsignals in NIH3T3 fibroblasts. Oncogene 1997: 14:2079–2089.

63. Rahman I, Gilmour P S, Jimenez L A, MacNee W.Oxidative stress and TNF-alpha induce histone acetyl-ation and NF-kappaB/AP-1 activation in alveolarepithelial cells: potential mechanism in gene transcrip-tion in lung inflammation. Mol Cell Biochem 2002:234–235: 239–248.

64. Zhou J, Jin Y, Gao Y et al. Genomic-scale analysis ofgene expression profiles in TNF-alpha treated humanumbilical vein endothelial cells. Inflamm Res 2002: 51:332–341.

65. Reenstra W R, Yaar M, Gilchrest B A. Effect of donorage on epidermal growth factor processing in man. ExpCell Res 1993: 209: 118–122.

66. Herlyn M, Berking C, Li G, Satyamoorthy K. Lessonsfrom melanocyte development for understanding thebiological events in naevus and melanoma formation.Melanoma Res 2000: 10: 303–312.

67. Park H Y, Perez J M, Laursen R, HaraM, Gilchrest B A.Protein kinase C-beta activates tyrosinase by phos-phorylating serine residues in its cytoplasmic domain.J Biol Chem 1999: 274: 16470–16478.

68. Freshney R I. Melanocytes. In: Frshney, R I, ed.Culture of animal cells. New York: John Wiley &Sons, 378–382.

Kim et al.

632

69. Gilchrest B A, Friedman P S. A culture system for thestudy of human melanocyte physiology. In: Jimbow K,ed. Structure and function of melanin. Sapporo, Japan:Fuji-shoin, 1987: 1–13.

70. Gilchrest B A, Vrabel M A, Flynn E, Szabo G. Selectivecultivation of human melanocytes from newbornand adult epidermis. J Invest Dermatol 1984: 83:370–376.

71. Resink T J, Scott-Burden T, Weber E, Buhler F R.Phorbol ester promotes a sustained down-regulation ofendothelin receptors and cellular responses to

endothelin in human vascular smooth muscle cells.Biochem Biophys Res Commun 1990: 166: 1213–1219.

72. Kock A, Schwarz T, Kirnbauer R et al. Humankeratinocytes are a source for tumor necrosis factoralpha: evidence for synthesis and release upon stimula-tion with endotoxin or ultraviolet light. J Exp Med1990: 172: 1609–1614.

73. Sondell M, Lundborg G, Kanje M. Vascular endothelialgrowth factor stimulates Schwann cell invasion andneovascularization of acellular nerve grafts. Brain Res1999: 846: 219–228.

Modulation of VEGF receptors

633