Copyright # Blackwell Munksgaard 2003

Experimental Dermatology 2003: 12 (Suppl. 2): 28–34
Blackwell Munksgaard . Printed in Denmark

EXPERIMENTAL DERMATOLOGYISSN 1479-1250

Differential expression of heparin-binding
EGF-like growth factor (HB-EGF) mRNAin normal human keratinocytes induced bya variety of natural and synthetic retinoids
Yoshimura K, Uchida G, Okazaki M, Kitano Y, Harii K. Differential
expression of heparin-binding EGF-like growth factor (HB-EGF) mRNAin normal human keratinocytes induced by a variety of natural andsynthetic retinoids.
Exp Dermatol 2003: 12 (Suppl. 2): 28–34. # Blackwell Munksgaard, 2003
Abstract: It was recently revealed that epidermal growth following topicaltreatment with all-trans retinoic acid (atRA) was at least partly induced byheparin-binding epidermal growth factor-like growth factor (HB-EGF)
released from suprabasal keratinocytes. Since proliferation of
keratinocytes appears to be one of the critical roles of atRA in
depigmentation treatment and promotion of wound healing, HB-EGF isconsidered suitable for assessing the therapeutic value of topical retinoids.
In this study, HB-EGF mRNA expression in normal human keratinocytes
after atRA treatment was examined, and the effects of a variety of naturaland synthetic retinoids were compared. The results of reverse
transcription polymerase chain reaction (RT-PCR) suggested thatinduction of differentiation increased HB-EGF mRNA expression in
cultured keratinocytes. Real-time PCR analyses revealed that HB-EGF
mRNA expression was elevated dose-dependently with atRA, peaking at
12 h. This elevation was more prominent in confluent keratinocytes thanin subconfluent cells, suggesting that differentiated keratinocytes are more
subject to stimulation of HB-EGF expression by atRA than proliferating
keratinocytes. HB-EGF mRNA was upregulated in differentiationinduced keratinocytes by all retinoids used in this study at 1 mmol/l, andmarked upregulation was seen when treated with three isotypes of retinoic
acid (atRA, and 9-cis and 13-cis retinoic acid). RARa-selective agonists(Am80, Am580, ER-38925, and TAC-101) and a panagonist of RARs
(Re80) caused relatively low elevation of HB-EGF transcripts, as did
all-trans retinol (Rol) and all-trans retinal (Ral). Although anotherpanagonist (Ch55) showed the highest elevation of HB-EGF mRNA, it
was relatively cytotoxic at the concentration employed. Ral and Rol were
found to upregulate HB-EGF when used at 100 mmol/l to 1 mmol/l, to asimilar extent of atRA at 1–10 mmol/l. The capacity of retinoids toupregulate HB-EGF may be an important index for investigation anddevelopment of an ideal synthetic retinoid, which has maximum benefits
and minimum side-effects

Introduction
Since the 1970s, topical tretinoin (all-trans retinoic
acid; atRA) has been widely used for several skindiseases such as acne vulgaris and photoaged skin,with remarkable success. Retinoids have a varietyof biological effects on the epidermis and dermis
including skin appendices (such as hair follicles

28

Kotaro Yoshimura, Gentaro
Uchida, Mutsumi Okazaki,
Yukie Kitano and Kiyonori Harii
Department of Plastic and Reconstructive
Surgery, Graduate School of Medicine, Universityof Tokyo, Tokyo, Japan.

Keywords: heparin-binding EGF-like growth
factor (HB-EGF) – keratinocyte – retinoid –retinol – tretinoin
Kotaro Yoshimura MD
Department of Plastic and Reconstructive
Surgery
Graduate School of Medicine
University of Tokyo
7-3-1 Hongo
Bunkyo-ku
Tokyo 113-8655
Japan.
Tel: þ81 3 5800 8670
Fax: þ81 3 5800 6929
E-mail: yoshimura-pla@h.u-tokyo.ac.jp

and sebaceous glands), which are mediated by specific nuclear receptors, the RARs (retinoic acidreceptors) and RXRs (retinoid X receptors) (1,2).
Epidermal hyperplasia is one of the most prominent
histological changes in skin seen after treatment
with atRA (1,3). This phenomenon has been commonly observed not only in vivo in several mammals, but also in skin equivalents using normal

Differential expression of HB-EGF mRNA by retinoids

human keratinocytes (NHK) and fibroblasts (datanot shown). However, since NHK proliferation was
not consistently observed in monolayer-cultured
NHK (2,4), the mechanism of keratinocyte proliferation induced by retinoids remained unknown fora long time.
Recently, a study using cultured keratinocytes,organ culture and skin biopsies revealed that transcripts of heparin-binding epidermal growth factorlike growth factor (HB-EGF), a member of the
EGF family of growth factors, are induced by treatment with retinoids, suggesting that epidermal
hyperplasia after atRA treatment may be mediated,
at least in part, by keratinocyte-derived HB-EGF(5). HB-EGF has been shown to be upregulated in
actual wound healing far more than other growth
factors that accelerate epidermal growth (6). Since
then, a paracrine action of HB-EGF released fromsuprabasal keratinocytes has been found to be a key
mechanism of epidermal growth following atRA
treatment, in a study using transgenic mice (7).
Thus, it is suggested that atRA accelerates keratinocyte differentiation directly, and promotes keratinocyte proliferation indirectly.
Combination topical therapies with atRA andhydroquinone for pigmented skin lesions have beensuccessfully performed since Kligman and Willis
proposed their depigmenting formula in 1975 (8).
The authors modified the protocol and demonstrated the depigmenting potential of atRA (9,10).
Since atRA appeared not to have a suppressive effecton melanogenesis, keratinocyte proliferation andacceleration of epidermal turnover (keratinocyte differentiation) appear to be the two critical effects ofatRA in the depigmenting therapies (11). Since theformer is mediated by a paracrine action of HB-EGF
released from suprabasal keratinocytes, HB-EGF
mRNA is thought to be suitable for assessing thetherapeutic value of topical retinoids in treating
hyperpigmentation or promoting wound healing.
The purposes of this study are to reconfirm theeffect of atRA in promoting the expression of HBEGF mRNA in normal human keratinocytes, andto compare the HB-EGF-promoting abilities of avariety of natural and synthetic retinoids. The
capacity of retinoids to upregulate HB-EGF may
be an important index for investigating and developing an ideal synthetic retinoid, which has maximum beneficial effects and minimum side-effects.
Methods
Cell isolation and cell culture
Human epidermal keratinocytes isolated from biopsies of healthyskin obtained from young Japanese patients during plastic surgery were used in this study. Keratinocytes were isolated using amodification of the method reported previously (12). Briefly, thespecimens were washed three times in phosphate buffered saline(PBS) and finely shredded with scissors, and incubated with
0.25% trypsin and 0.02% EDTA in PBS for 16–24 h at 4 C.
The epithelium was separated from the dermis with forceps, andkeratinocytes were isolated from the subepithelial side. Keratinocytes were grown in a modified serum-free keratinocyte growth
medium (KGM; Kyokuto Seiyaku, Tokyo), which consists of
MCDB153 with high concentrations of amino acids, transferrin(final concentration 10 mg/ml), insulin (5 mg/ml), hydrocortisone(0.5 mg/ml), phosphorylethanolamine (14.1 mg/ml), and bovine
pituitary extract (40 mg/ml). The final concentration of Ca2þ inthe medium was 0.03 mmol/l.
Keratinocytes in a subconfluent state (30% confluency) or
those in confluent state (cultured for 48 h after they showed
100% confluency) were used in this study, representing growing
and differentiated keratinocytes, respectively.

Reagents
The influence of various kinds of retinoids on HB-EGF mRNA
expression of keratinocytes was compared in this study. Fivenatural retinoids and six synthetic retinoids were used (Fig. 1).
The natural retinoids were tretinoin (all-trans retinoic acid;atRA), 13-cis retinoic acid (13cRA), 9-cis retinoic acid (9cRA),
all-trans retinol (Rol), and all-trans retinal (Ral), all purchasedfrom Sigma (St Louis, MO, USA). 9cRA is known to be a ligand
for RXRs and also binds to RARs. The synthetic retinoids were
Am80 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-carbamoyl] benzoic acid), Am580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido] benzoic acid), Ch55 ((E)-4[3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl] benzoic acid), Re80
(4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl- 2-naphthalenyl)-1-propenyl] benzoic acid), TAC101 (4[3,5-bis (trimethylsilyl) benzamide] benzoic acid) and ER-38925(4-[5-(4,7-dimethylbenzofuran-2-yl)pyrrol-2-yl] benzoic acid). All
six retinoids were generous gifts from Dr Kagechika (Universityof Tokyo, Tokyo) (Am80, Am580, Ch55 and Re80), Taiho Pharmaceuticals Co, Ltd (Tokyo, Japan) (TAC101) and Eisai Co, Ltd.
(Tokyo, Japan) (ER-38925). Am80, Am580, TAC-101, andER-38925 are RARa-selective agonists (13–16), while Ch55 andRe80 are panagonists for all three RAR subtypes (12,17).
All reagents were dissolved in ethanol at 1 mmol/l as stock
solutions (for atRA, Rol, and Ral; other stock solutions werealso prepared), and 10 ml of each stock solution was added to
10 ml of the culture medium to get the designated final concentrations (for comparison of all retinoids; 1 mmol/l). As a control,
10 ml of ethanol alone was added to 10 ml of the culture medium.

RNA isolation
After removing the culture media and washing twice with PBS,
total RNA was obtained with RNeasy1 Mini Kit (Qiagen,Hilden, Germany). In order to eliminate residual genomic DNA,a RNase-Free DNase Set (Qiagen) was then used. The concentration of each RNA sample was measured with a spectrophotometer (V-530 UV/VIS; Jasco, Tokyo, Japan).

Reverse transcription-polymerase chain reaction
analysis
The amount of isolated total RNA was spectrophotometricallymeasured. A reverse transcriptase-polymerase chain reaction(RT-PCR) was performed using RNA PCR Kit (AMV, Ver. 2.1;
TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions; 5 mg of total RNA in 100 ml/l of reaction mixture (final
concentrations: 5 mmol/l MgCl2, 1 mmol/l dNTP mixture, 1 U/ml
RNase inhibitor, 0.125 mmol/l oligo dT-Adaptor primer, 10 mmol/l
Tris-HCl, 50 mmol/l KCl, pH 8.3) containing 25 U of AMV

29

Yoshimura et al.

Figure 1. Chemical structures of retinoids used in this study. Left panel: five natural retinoids. atRA: all-trans retinoic acid, 13cRA:
13-cis retinoic acid, 9cRA: 9-cis retinoic acid, Rol: all-trans retinol, Ral: all-trans retinal. Right panel: six synthetic retinoids.
Reverse Transcriptase XL at 42 C for 30 min, followed byinactivation of the enzyme at 99 C for 5 min with Program
Temp Control System (PC-700; Astec, Fukuoka, Japan). The
control reaction was performed simultaneously under identicalconditions, but without reverse transcriptase.
For PCR amplification, 0.5 ml of each cDNA reaction mixture
was added to 49.5 ml PCR mixture, containing 5 ml MgCl2(25 mmol/l), 5 ml 10 PCR buffer, 1 ml deoxynucleotide mixture(10 mmol/l), 0.5 ml each of the 30 and 50 primers (50 mmol/l each),
0.25 ml Taq polymerase, and 37.25 ml DEPC-treated water. Reaction mixtures were amplified using a thermal cycler (Microplate
Gradient PC-960G; Corbett Research, Australia). The PCR cycle
conditions were: melting for 30 s at 94  C, annealing for 30 s at
59  C, and extension for 1.5 min at 72  C. The oligonucleotide
primers used for RT-PCR were as follows; human HB-EGF (forward) 50 -CACACCAAACAAGGAGGAGC-30 and (reverse)
50 -CATGAGAAGCCCCACGATGA-30 (PCR-product size 279bp); human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(forward) 50 -GAAGGTGAAGGTCGGAGTC-30 and (reverse)
50 -GAAGATGGTGATGGGATTTC-30 (PCR-product size 226bp). All RT-PCR products were separated on 2% agarose gels,visualized by ultraviolet B using ethidium bromide staining.

Real time RT-PCR analysis
Expression of HB-EGF transcripts by keratinocytes wasquantitatively measured using a real-time quantitative PCR

30system (Sequence Detection System ABI PRISM 7700; PE
Applied Biosystems, Foster City, CA, USA). Real-time PCR
was performed on 96-well optical reaction plates (ABI
PRISMTM; PE Biosystems). All PCR reaction mixtures contained per well 25 ml of TaqMan SYBR1 Green PCR MasterMix (PE Biosystems), 0.25 ml of forward primer (10 pmol/ml),
0.25 ml of reverse primer (10 pmol/ml), 4 ml of each diluted
sample (RT products), and 20.5 ml of distilled water. PCR
amplification of each sample was performed with both specific primer pairs of target human HB-EGF gene and human
GAPDH gene on the same reaction plate. The oligonucleotide
primers used for real-time PCR were as follows: human
HB-EGF (forward) 50 -CAGATCTGGACCTTTTGAGAGTCA-30
and (reverse) 50 -TCCCGTGCTCCTCCTTGTT-30 (PCR-productsize 78 bp); human GAPDH (forward) 50 -GAAGGTGAAGGTCGGAGTC-30 and (reverse) 50 -GAAGATGGTGATGGGATTTC-30 (PCR-product size 226 bp). The PCR
reaction comprised 40 cycles; denaturing at 95 C (15 s),annealing/extension at 60 C (1 min). For comparison with
the HB-EGF mRNA expression, the value of HB-EGF ofeach sample was normalized by deducting the value of
GAPDH from the same sample. In order to eliminate thepossibility of contamination of genomic DNA during extraction of total RNA, a control reaction with each primer pair
was performed at the same time under identical conditions
without reverse transcription, and no amplification was
detected. The normalized data were collected from at least
four separate experiments.

Differential expression of HB-EGF mRNA by retinoids

Statistical analysis
In each experiment, the values of normalized HB-EGF mRNA
expression of each sample were divided by the value of the control
to obtain data for analysis. Statistical analysis was performed using
Student’s t-test. A value of P < 0.05 was considered significant.

Results
RT-PCR analysis
In preliminary experiments, HB-EGF mRNA
expression was examined by RT-PCR. HB-EGF
mRNA expression was elevated relative to controls in keratinocytes that had been induced todifferentiate by incubation for 48 h with high concentrations (1.8 mmol/l) of Ca2þ or 2% serum, orboth (Fig. 2). This finding suggested that normal
human keratinocytes increase HB-EGF mRNA
expression when they differentiate.
The investigation of sequential changes of HBEGF mRNA expression by atRA (1 mM) revealed
that HB-EGF mRNA expression peaked at 12 hafter atRA administration (Fig. 3). In addition,
our results also showed that HB-EGF transcripts
were dose-dependently elevated by incubation
with atRA for 15 h in the concentration range of
0.01–1 mmol/l (Fig. 4).
HB-EGF mRNA elevation by atRA in subconfluent
or confluent keratinocytes
The sequential changes of HB-EGF mRNA elevation by atRA (1 mmol/l) was quantitatively measured with real-time PCR using normal humankeratinocytes in subconfluent (30% confluence)and confluent (100% confluence) states (Fig. 5).
For confluence, keratinocytes cultured for 48 h
after they were first observed to be confluent inculture dishes were used, representing differentiated keratinocytes, as a model of suprabasal keratinocytes. For subconfluence, keratinocytes showing 30% confluency were used immediately,

Figure 2. HB-EGF mRNA expression in keratinocytes
differentiation-induced by 48 h incubation with medium
containing 2% serum or/and 1.8 mmol/l Ca2þ. It was amplified
by RT-PCR and is shown here with the control incubated withnormal medium. CTL: control, S: medium with 2% serum, Ca:medium with 1.8 mmol/l Ca2þ, S þ Ca; medium with both 2%
serum and 1.8 mmol/l Ca2þ.

Figure 3. HB-EGF mRNA expression in keratinocytes cultured
with medium containing 1 mmol/l atRA for 0, 6, 12, 24, and 48 h.
The expression was amplified by RT-PCR.

representing proliferating keratinocytes, as amodel of basal keratinocytes. The normalized
HB-EGF mRNA level was highest at 12 h, andthereafter decreased in both cases. Throughout
the time investigated (0–48 h), the average valueof HB-EGF mRNA level was higher in confluent
than in subconfluent keratinocytes. Statistical significance between the subconfluent and confluent
states was found only at 12 h.
Comparison of HB-EGF mRNA upregulation by
natural and synthetic retinoids
The HB-EGF mRNA levels were quantitatively
measured with real-time PCR in normal humanconfluent-state keratinocytes cultured for 15 hwith 11 retinoids (0.01 mmol/l, 0.1 mmol/l, and1 mmol/l atRA; 1 mmol/l for the other retinoids).
The results demonstrated that all 11 retinoids studied showed significant upregulation of HB-EGF
mRNA, but to different extents (Fig. 6). Ch55, apanagonist of RARs, demonstrated a much higher
degree of upregulation of HB-EGF transcripts
than the three retinoic acids, but the amount of
total RNA isolated from keratinocytes treatedwith Ch55 was very small compared with thatfrom the other samples, suggesting that Ch55 wascytotoxic to keratinocytes in the concentration
employed here (1 mmol/l). Three isotypes of retinoic acid (atRA, 9cRA, and 13cRA) showed
higher degrees of upregulation of HB-EGF transcripts than any other natural or synthetic

Figure 4. HB-EGF mRNA expression in keratinocytes cultured
with medium containing rhree concentrations (1 mmol/l,
0.1 mmol/l, and 0.01 mM) of atRA for 15 h. The expression was
amplified by RT-PCR.

31

Yoshimura et al.

was significantly more upregulated by atRA thanby Rol or Ral (Fig. 7). When used at concentrations of 100 mmol/l to 1 mmol/l, atRA was cytotoxic, whereas Rol and Ral markedly upregu lated
HB-EGF to a similar degree as atRA at
1–10 mmol/l.
Discussion
Figure 5. HB-EGF mRNA expression in keratinocytes insubconfluent (30%) or confluent (100%) states cultured withmedium containing 1 mmol/l atRA for 0, 6, 12, 24, and 48 h. HBEGF mRNA was measured quantitatively with real-time PCRsystem and the normalized value was calculated by deducting the
GAPDH value of each sample. The values are demonstrated as
relative values to the value obtained from the subconfluent
keratinocytes at 0 h, and indicated as mean + standard error.
* P < 0.05.

retinoids except Ch55, although there was noapparent difference among the three retinoic
acids. RARa-selective synthetic agonists (Am80,
Am580, ER-38925, and TAC-101) and anotherpanagonist of RARs (Re80) showed a lesser
degree of upregulation of HB-EGF transcripts
than the three retinoic acids, as did the othertwo natural retinoids, Rol and Ral.
Comparison of upregulations of HB-EGF mRNAby atRA, Rol and Ral
HB-EGF transcript expression in normal humanconfluent keratinocytes treated for 15 h with several concentrations of atRA, Rol and Ral were
quantitatively measured using real-time PCR.
Compared at 1 mmol/l and 10 mmol/l, HB-EGF

Figure 6. HB-EGF mRNA expression in normal humanconfluent keratinocytes cultured for 15 h with 1 mmol/l of eachretinoid (0.01, 0.1, 1 mmol/l for atRA). HB-EGF mRNA wasmeasured quantitatively with real-time PCR system. The values
are demonstrated as relative values to the value obtained fromthe control in each measurement, and indicated as mean +standard error. CTL: control, atRA (6): 1 mmol/l atRA, atRA
(7); 0.1 mmol/l atRA, atRA (8): 0.01 mM atRA, Er: ER-38925,
TAC: TAC-101. The results of all retinoids are significantly
higher than that of control.

32

It was recently revealed that HB-EGF mRNA canbe induced by treatment of retinoids in humankeratinocytes and organ-cultured skin, suggestingthat epidermal hyperplasia after atRA treatment
may be mediated, at least in part, by keratinocytederived HB-EGF (5). Thereafter, a paracrine
action of HB-EGF released from suprabasal keratinocytes was suggested to be a key mechanism of
epidermal growth following atRA treatment in the
study, in which dominant-negative RARa wasoverexpressed in suprabasal layers of mice, andthe response of skin to atRA was examined (7).
The present study confirmed that HB-EGF
mRNA is markedly induced by atRA in humannormal keratinocytes, and suggests that even celldifferentiation of keratinocytes alone might
increase keratinocyte-derived HB-EGF without
retinoid treatment. Although the previous study(5) did not detect a significant difference in HBEGF upregulation between confluent and subconfluent keratinocytes at 6, 24 or 48 h, our results
revealed that differentiated confluent keratinocytes upregulate HB-EGF significantly more thangrowing subconfluent keratinocytes after 12 h of
atRA treatment, supporting the hypothesis of

Figure 7. HB-EGF mRNA expression in normal humankeratinocytes in confluent condition cultured for 15 h with oneof the three natural retinoids (atRA, Rol, and Ral). HB-EGF
mRNA was measured quantitatively with real-time PCR system.
The values are demonstrated as relative values to the value
obtained from the control (normal medium) in each measurement.
Four concentrations (0.01 mmol/l, 0.1 mmol/l, 1 mmol/l, and
10 mmol/l for atRA; 1 mmol/l, 10 mmol/l, 100 mmol/l, and 1 mmol/lfor Rol and Ral) were employed for each measurement. The
values are indicated as mean + standard error. At theconcentration of 1 mmol/l and 10 mmol/l, HB-EGF mRNA
expression was significantly higher in keratinocytes treated with
atRA than those treated with Rol or Ral. * P < 0.05.

Differential expression of HB-EGF mRNA by retinoids

Xiao et al. (7) that HB-EGF released from differentiated suprabasal keratinocytes stimulates proliferation of basal keratinocytes. Whether the
induction of HB-EGF mRNA is due to liganddependent transcription activation of the HBEGF gene or not is to be explored, because thepresence of retinoic acid response elements(RARE) has not yet been identified in the promoter region of the HB-EGF gene (5,7).
As far as depigmenting treatment is concerned,
promotion of keratinocyte proliferation and acceleration of keratinocyte differentiation appeared tobe the two main roles of atRA (11). Assuming that
HB-EGF released from suprabasal keratinocytes
is the main reason for epidermal growth following
retinoid treatment in vivo, the promotive ability ofretinoids on HB-EGF expression can be used as anindex for evaluating individual retinoids and developing the synthetic retinoids to promote epidermal
growth. However, a reliable index for indicating
the ability of retinoids to differentiate keratinocytes has not been found.
All the natural and synthetic retinoids employedin this study significantly upregulated HB-EGF,
suggesting that epidermal growth can be induced
by a wide range of topical retinoids. However, theextent of HB-EGF upregulation was distinct
among those agents. All four RARa-specific synthetic retinoids, Am80, Am580, ER-38925, andTAC-101, showed relatively low promotion of
HB-EGF mRNA compared with all three isotypesof retinoic acid, whereas they demonstrated ahigher affinity to RARa and higher activity inother actions such as growth suppression of neoplasm than did atRA (13–16). The reason for thevariation in promotion of HB-EGF mRNA among
retinoids remains unknown, but it may be partly
due to differential binding affinity to RARg. Ninety
percent of the RARs expressed in epidermis are
RARg, and the others are RARa, while RXRa isa major RXR in skin (2,18). Both RARs and RXRs
were reported to have higher expression in the
spinous and granular layers compared with thebasal layer in normal skin (19). Since RAR-b- or
RAR-g-specific ligands were not evaluated in thisstudy, the relationship between retinoid specificity
to subtypes of RARs and HB-EGF expression
requires further investigation.
In addition to mediating RARE- or retinoid X
receptor responsive elements-dependent transactivation, retinoid receptors can also affect gene
expression by inhibiting the action of other transactivation factors, including AP-1 (20). Furthermore,
there are some RAR/RXR-independent pathways,
such as activation of the mannose-6-phosphate(M6P)/insulin-like growth factor-II (IGF-II) (21).

Human keratinocytes are known to transform
Rol into Ral, and then into atRA by two enzymatic steps involving dehydrogenases, and theconversion rate of Rol into atRA depends on thestate of keratinocyte differentiation; differentiatedkeratinocytes can convert at a higher rate thannon-differentiated keratinocytes (22). The binding
affinity of Rol or Ral to retinoid nuclear receptors
is quite low (23), so that their biological activity
should result from their oxidative transformation
into retinoic acid by epidermal keratinocytes.
However, there have been some reports suggesting
the existence of other pathways than that mediatedby nuclear receptors (24).
Rol is constitutively present in human plasma at
1–2 mmol/l (25) and the upper limit of extracellular
Rol concentration in epidermis is thought to be
0.7 mmol/l (26), whereas atRA is present at 4–
14 nmol/l in human plasma (27,28). The present
results revealed that atRA, Rol and Ral all markedly induce HB-EGF mRNA at pharmacologicalconcentrations, which are 100–1000-fold higher
than physiological concentrations. Rol and Ral
were in fact able to induce high levels of HBEGF mRNA when used at much higher (40–100
fold) concentrations than atRA. This means that
Rol and Ral, when used at high concentrations,
could promote epidermal growth in vivo like atRA,
although Rol and Ral have been reported to show
far fewer side-effects than atRA when used atsimilar concentrations. Rol and Ral are thought
to be more tolerable because of less extensive sideeffects, and may be of value in clinical use.
The results indicated the potential for use of Rolor Ral at high concentrations in vivo, especially indepigmenting treatments, which need promotionof epidermal growth and turnover. Indeed, ourpreliminary clinical study with 5–10% Rol aqueous gel suggested similar depigmenting effects to
0.1% atRA aqueous gel (29). Thus, promotion of
HB-EGF expression may be used as a promisingindex for evaluating the depigmenting ability of
topical retinoids.
Acknowledgements
We thank Dr Hiroyuki Kagechika (University of Tokyo,
Tokyo, Japan) for his generous provision of Am80, Am 580,
Ch55, and Re80. We also acknowledge Yuka Kuwahara forher technical assistance.

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