British Journal of Dermatology 1996; 134: 4 3 1 - 4 3 6 .
Topical retinoic acid changes the epidermal cell surface
glycosylation pattern towards that of a mucosal epithelium
C.E.M.GRIFFITHS. E.DABELSTEEN* AND J.j.VOORHEES
Department of Dennatologn, University ofMichiijan Medical Center, Ann Arbor, Michiijan. U.S.A.
*School of Detitistrii. Department of Oral Diafimstii's. Copenhaijen. Denmark
Accepted for publication 26 May 1995
Summary
Topical all-trims retinoic acid (RA) produces a nutnber ol epidermal changes which are indistinguishable l'rom those observed following treatment with a local irritant, namely sodium lauryl sulphate
(SI.S). Thisdhserviition has led to criticism thai the efficacy of RA in disorders such as photoageing. Is
merely a result of irrilancy. In stratitied epithelia. the cellular difterentiation process is characterized
by a stepwise synthesis of cell surface carhohydrates. and each type of stratitied epithelium has its
own specific pattern of carbohydrate expression. Glycosyltrimsferases. which are responsible for
carbohydrate synthesis, are influenced by retinoids. Thus, we investigated whether epidermal cell
surface glycosylation is altered in skin treated with topical RA, and contrasted it wilh changes
induced by topical SLS.
Skin biopsies were obtained from seven normal volunteers who had been treated, on three
separate areas of buttock skin, with single applications of 0 1% RA. 2% SLS, or vehicle creatiis,
followed by 4-day occlusion. Biopsies were assessed imniunohistologically using highly specific
monoclonal antibodies lo cell surface carbohydrates (types 1, 2 and 3 chain structures), previously
demonstrated in the epidermis and iti oral mucosal epithelium. Although type 1 chain structures
were not demonstrated in any of the samples, the distribution of type 2 and 3 chain structures in RAtreated epidermis was altered towards that seen in a mucosal epithelium. T antigen, a mucin-type
cell surface carbohydrate structure normally expressed throughout the epidermis, was only observed
in the granular layer of RA-treated epidermis—a feature of mucosal epitheiia. Le^, normally only
seen in non-keratinized buccal epithelium, was strongly expressed in RA-treated epidermis. In
contrast, the glycosylalion pattern of the SLS-treated epidermis was tiot significantly different frotn
that observed after vehicle treatment. Thus, RA treattnent converts normal stratified epithelium
towards the phenotype of mucosal epitheliutn with a decrease in T antigen and a concomitant
increase in Le^. These changes iire not observed following treatment with SLS and identify an
itnportant difference between RA elTects and irritancy.
Topical ttll-trans retinoic acid (RA) is efficacious for the
treattnent of various skin disorders, including acne' and
photoageing." It is accepted that RA is beneficial in
acne due to the nortnalizatioti of follicular duct
keratitiocyte differentiation. However, the mechanism
of action of RA in the improvement of clinical features of
photoageing, namely wrinkling and aclinic lentigines,
is still controversial. This situation has arisen because
the cutaneous erythema and scaling (retinoid
dermatitis), associated with the use of topical RA,
raises the possibility that RA is acting solely as a local
irritant and that its actions are not an inherent property
Correspondence: Professor C.F,.M.("!rifflths. Section of Dermatology.
tiniversity of Manchester School of Medicine. Hope Hospital. Salford
M6 8HD. U.K.
:r 199f> British Association of Dermatologists
of the RA molecule. In addition, 4~day treatment of
normal skin with RA, under occlusion, produces
epidermal histological changes very similar to those
produced by sodium lauryl sulphate (SLS) under the
same conditions.' However. RA produces changes not
seen in SLS-treated skin. e.g. the induction of cellular
retinoic acid binding protein II (CRABP-II)"* and the
induction of RA 4-hydroxylase activity, both of which
are important in RA metabolism.
At the cell surface, monosaccharides are linked
together in different sequences by different glycosidic
linkages, thereby generating a vast number of complex
saccharide chains'' ' (Fig. 1). In the stratified epithelia of
skin and oral mucosa. the cellular differentiation
process is characterized by a stepwise synthesis of cell
431
432
C.E.M.GRIFFITHS etai
Type 1 chain
precursor
H type 1 chsin
I
FOCdi
Gal|3I-3GlcNAclJ1-R
G3l31-3GlcNAcBi-R
2
I
FuCili
FuCul
4
Fuca1
Fucol
NeuAc2
Fucul
Tn
H type 3 chain
-3GalN Acn 1 -O- SerH^hr
3GalNAci.1-0-Sermir
Gal01 -3GalNAcii 1 -O-
I
Sialosyl-T
NeuAcii
2
Siaiosyl-Tn
NeuAca
2
NeuAcn
2
Galpi •3GalNAc<.1 -O-Ser/Thr
surface carbohydrates.^'' The different types of stratitied
epithelium have their own specilic pattern of carbohydrate expression, which may vary in relationship to
certain physiological and pathological conditions.'" '"^
II appears that RA-treated epidermis takes on some of
the characteristics of mucosal epithelia, i.e. glycosaminoglycan accumulation '' and the expression of
keratin 13. *' Expression of glycosyltransferases. which
are responsible for the synthesis of cell surface carbohydrate chains, is changed in vitro by RA,'' indicating
that RA may ultimately regulate the expression of cell
surface carbohydrates. In the light of these observations, and in an attempt to further characterize the
differences betweeti RA and SLS-trealed skin, we examined the ability of these two compounds to modulate the
expression of epidermal cell surface carbohydrates, i.e.
the glycosylation pattern.
Materials and methods
Pttticnts
Seven normal volunteers (age range 3 5-45 years) were
treated on three separate areas of the hips or buttocks
with single applications of ()-l% RA cream (Retin-A-,
Ortho Pharmaceutical Corp., Raritan. NJ. U.S.A.)
G a l N A c i O-SeriThr
Figure 1. Biosynthetic pathway for types 1, 2, and 3
chain Ciirbohydrate antigens. .Arrows indicate the
synthetic SLHitience of these structures. Types 1 and
2 chain precursors may be carried either on
glycoprolt'ins or (jlycolipids. whereas type 3 chain
structures are 0-linked carbohydrate chains hound
to serine or threonine. Each biosynthetic step is
catalysed by a specilic enzyme: type 1 chain
structures are found exclusively in mucosnl
epithelium, whereas type 2 and J chains may IK?
found in mucosal as well as epitiermni cells.
colour-matched, vehicle cream (Ortho), or 2% SLS
(Dupouol-C, Dupont Co., Wilmington. DE, U.S.A.)
formulated in Retin-A vehicle cream. The treated areas
were occluded with light-tighl plastic for 4 days. We
havepreviously demonstrated lhal()-l%RA and 2% SLS
creams produce almost identical erythema and epidermal histological changes under these treattnent conditions.' At 4 days, the occlusion was removed and 4-tnm
punch biopsies taken from all treatment sites under 1%
lignocaine anaesthesia. The bi<jpsies were snap frozen in
liquid nitrogen and stored at - 7 0 C until use.
All volunteers gave written consent and the protocol
was approved by the University of Michigan Medical
Center Institutional Review Board.
Immtmochemistrij
Five-micrometer cryostat sections were air-dried and
stained using a double-layer technique'" with wellcharacterized monoclonal antibodies to carbohydrate
epitopes (Table 1).'^""' In brief, frozen sections were
air-dried and incubated in a moist chamber with a
monoclonal antibody lor 1 S h at 4 C. Following ihree
washes in phosphate-buffered saline (PBS) pH 7 2
(5 min each), fiuorescein isothiocyanate (FITC)conjugated rabbit anti-mouse immunoglobulin, diluted
1996 British Association of Dermatologtsts, British Journal of Dermatology. 134, 4 3 1 - 4 3 6
RETINOIC ACID CHANGES GLYCOSYLATION
Tnhk- I. I'iini'l of mouse monoclontil antibotlics used
Antlbody/lsotype
Antigen
Source (reference)
IA:"
(IS)
Chcmbiomcd, Ciinada
(lyi
(19)
(20)
(21)
(22)
DAKO, Denmark
DAKO. Uenmark
(23)
433
(granular and upper spinous): and 3. >3 cell layers
stained.
Stdtistical inelJiods
CF4-C/lgG
Anti-Lc''/IgM
1 B2/lgM
HK2/lgM
Slll/lgC,
AHfVlgM
FIKVIgM
llB-'i'n/IgM
1 IB-T/IgM
HHl4/lg.M
Le"
N-Lac
H
Le^
Le''
MsLe^ext.
Tn
T
H
1 :4() (Dakopatts. Copenhagen. Denmark), was applied
for 40min. The sections were again washed and
inouiilcd in I'BS containing j?-phenylene-diamine in
glycerol. All monoclonal antibodies were from hybridoma cuittire supernatant.s containing approximately
1 ()- i() |ig of immunogiobulin per millililre. In order to
unmask antigenic determinants, enzymatic deglycosylalion of sialic acid residues was performed by ncuraininidase treatment ol' additional sections before
staining, as described previously. In brief, sections
were incubaled with neuraminidase type 6 (Sigma
Chemical Co. St Louis. MO, U.S.A.) from Clostridiiim
IH-rfriiufens type 6 (Sigma). ()-l U of neuraminidase per
millilitre in a 0-lmol/l acetate, and ()-()4mol/l CaCU
huHer at pH S-S. The slides were examined, under epiillumination. using a Zeiss fluorescence microscope
equipped with a FITC interference filter and a 50-W
xenon lamp. Control reactions for ihe specificity of the
staining consisted of: |i) staining with conjugate alone:
(ii) substitution of the primary monoclonal antibody
with monoclonal antibodies of irrelevant .specificity hut
of the same isotype: and (iii) substitution of the primary
antibody with culture supernatant from the myeloma
line (Sp2/()| used for hybridization, so as to control for
the culture medium and nonspecific secretions from the
myeloma cell line.
The sign test was used to compare the presence or
absence of staining among treatments for T antigen as
well as I^e-^. The Friedman rank sum and multiple comparison tests were used to compare group differences iti
epithelial thickness. All P-values are two-sided.
Summary statistics are expressed as means ± standard
error of the mean (SEM). The data were analysed wilh
the Michigan hileractive data analysis system, a
statistical software package developed by the Center
for Statistical Consultation and Research at the
University of Michigan.
Results
As previously observed.' both RA and SLS treatments
thickened the epidermis. However, there was no
significant difference between the epidermal thickness
of RA and SLS-treated skin (Table 2).
Type 1 chain structures (Le" and Le ) could not be
demonstrated in vehicle-. SLS- or RA-treated skin. H
antigen, which is a precursor of Le^\ was not seen in
RA-treated tissue but was present in the granular cell
layer of vehicle- and SLS-treated skin, whereas
JV-acetyllactosamine. the precursor of H antigen, was
weakly expressed in all samples, Pretreatment with
neuraminidase showed strong staining in the spinous
cell layer, indicating that most of the N-acetyllactosamine
is masked by sialic acid. We could not demonstrate Le"
in any biopsies. However, sialylated Le'' was seen in all
biopsies, as cell surface staining on epidermal dendritic
cells, most probably Langerhans cells, as has been
Table 2. Effects of 4-day treatment wilh {)-\% retinoic acid (RAI. 2%
sodium Inuryl sulphate (SLS) or vehicle creams on epidermal
thickness, Ix."' ;iiul T antigen exprcssitm in normal skin
Histotogical parameter
Vehicle
{(I = 6)
(M'I'oKA
(" = ")
2% SLS
{n=7)
Epidermal thickness
T anligen
Le''
2-0 ± 0"
3 0 ± 0"
i)±(f
3i±()-5''
071 ± Oi''
21)11)4''
2-6 ± 0-2'*
J-0 ± 0*
()±()"
Quantification of staining
Epidermal thickness was graded from 2 (normal
thickness) to a maximum of fi. Epidermal T antigen
expression was graded on a 0 - 3 scale where 0 is
staining of 0 - 1 cell layers: 1. 2 - 4 layers: 2. > 4
hut not all cell layers: and 3, staining of entire
epidermis. Epidermal Le^ expression was also graded
on a 0 - 3 scale, where 0 is no staining: 1. 1 superficial
ceU layer (granular layer): 2. 2 - 3 superficial cefi layers
For scales used to estimate epidermal thickness. T antigen and IA^^
expression see text. All values are mean ± standard error of mean
(SEM).
"• ^ For each parameler, pairwise comparison of means wilh iit least
one superscript in common denotes non-signilicaut differences ill llie
O'OS level.
British Association ol' Dermatologists. British Journal of Dermatohgii, 1 54, 4 Jl-43f>
434
C.E.M.GRIFFITHS et al
described previously.^*'^^ Le*. a type 2 chain structure,
was not observed in either vehicle- or SLS-treated skin,
but was strongly expressed as keratinocyte cell surface
staining in the upper epidermis of RA-treated skin
(Table 2: Fig. 2). T antigen, a type 3 chain structure,
was expressed throughout the epidermis in vehicle- and
SLS-treated skin but was only observed in the granular
layer of RA-Lreated epidermis (P ^ 0 ()3. Table 2. Fig.
2).
All other carbohydrate structures investigated showed
no differences between RA-. SLS- and vehicle-treated
skin, and were distributed as described previously.**' '**
Discussion
Cell surface carbohydrates are of considerable interest
in studies of epithelial cells because these structures
vary markedly with important cellular processes such
as development, differentiation, and oneogenic
transformation.^"'"^ In stratified epithelium, basal cells
express short and unbranched carbohydrates whereas
suprabasal layers express carbohydrates with longer
structures.' Changes in expression of cell surface
carbohydrates may be due to changes in glycosyltransferase activity or to competition between different
glycosyltransferases for a cotnmon substrate.''' As
terminal portions of carbohydrates are strongly
inimunogenic. it is possible to raise antibodies useful
for their identification."*" Different carbohydrate
terminal structures are termed types 1. 2, 3 and 4."'
and these structures have different tissue distributions.
Types 1. 2. and 5 are found in non-keratinized
(mucosal) epithelium."^ whereas only types 2 and 3
are found in keratinized epithelium.
Previous
work ^ has shown a mucosal-!ike expression of
keratin, involucrin and epidermal transglutaminase in
RA-treated skin. i.e. expression of keratin 1 J and
precocious suprabasa! expression of involucrin and
epidermal transglutaminase. Thus, our current study
supports the concept of RA-lreated skin assuming the
phenotype of a mucosa! epithelium.
We studied three groups of carbohydrate structures
with well-known biosynthetic precursor product
relationships, including types 1. 2 and mucin-type 3
i'igure 2. Retinoic Hcid (RA) alters expression of cell surface carhohydrates in human epidermis. Vehicle, 2% sodium lauryl sulphate (Sl^) and 0-1%
RA creams were applied lopically to normal skin and occluded for 4 days. Sections from vehicle (a and d). SlU (h and el and R,'\ (cand f) treated skin
were stained with an immunolluorescence method to detect cell surface carbohydrates, (a-cl represent .sections stained with antibody to T antigen.
whereas I d-f) represent sections stained with antibody to Le*". Positive epidermal keralinocyte cell surface staining is idenliliable as green 'chickenwire', whereas non-specific, background staining, is yellow. In RA-treated epidermis, T antigen is only present on the mosi superllcial cells in tho
spinous cell layer (ci, whereas in vehicle (a) and SI,S-treated epidermis (b) T antigen is present on all spinous cells. U'^ is only expressed in RA-treated
epidermis (f | and is not expressed in either vehicle (d) or SLS-treated epidermis (e) (x5()).
1996 British Association of Dermatologists. British Journal of Dermatology. H 4 . 4 3 1 - 4 3 6
RETINOIC ACID CHANGES GLYCOSYLATION
chain, in skin treated with either RA. SLS or a nonirritant vehicle. We found that distribution of carbohydrale structures in SLS and vehicle-treated epidermis
corresponds to that seen in the normal epidertnis. Type
1 chain structures that are normally expressed only in
mucosal epithelium^ '"* were not found in any ofthe
skin samples. However, type 2 and 3 chains showed
some mucosa-iike expression in RA-treated skin. In
normal stratified epithelium there is a sequential expression of type 2 chain N-acetyllactosamine. H antigen and
bkH)d group A and B antigens in A- and B-positive
individuals.'^'" However, in RA-treated skin, the
epidermis expressed Le^ (Fig. 2), which is a fucosylsubsliliited version of the H antigen not present in
normal epidermis but strongly expressed in mucosal
epithelitim,'" ""^ H antigen, which is found in normal
epidertnis, could not be detected in RA-treated
epidermis. Aciivity of the enzymes required for
synthesis of H antigen and Le^ has previously been
demonstrated in normal epithelium.'' '" However, these
activities have been expressed in different cell layers
and. therefore, did not result in expression of Le-^ but in
two precursor structures." namely Le" and H (Fig. 2).
The present work demonstrates that RA-treated skin
has an alteration in the differentiation pattern, thereby
leading to expression of X and H enzymes in the same
cells, and leading to a new cell surface carbohydrate
structure, i.e. Le^.
T antigen is an O-Iinked. mucin-Iike. carbohydrate
structure. "
hi the nortnal epidermis. T antigeti is
found on spinous and. to some extent, basal cells.^ but
following RA treatment, it could only be demonstrated
in the granular cell layer. Attempts to demonstrate the
precursor structures of T antigen (Tn and sialosyl-Tn) in
the basal and spinous cell layers failed. We believe that
T antigen in basal and spinous cell layers in RA-treated
tissue is masked by branching and elongation of carbohydrate structures, thereby leading to a mucin-type
glycosylation."' an observation which would be in
agreement with other studies indicating a mucous
type of differentiation after retinoid treatment.''^^ This
hypothesis is supported by studies ofthe effects of RA on
a mouse teratocarcinoma cell line. F9. ' Such studies
have shown that RA treatment induces differentiation
of F^J cells into primitive endoderm-iike cells, associated
with upregulation of N-acelylglucosamitie-tratisferases
which are responsible for synthesis of cell surface
O-Iinked polylactosamine. A polylactosatnine elongation t)f T antigen will block tissue staining with anti-T
antibodies, and could lead to some of the changes in
cell surface carbohydrates observed in RA-treated
435
skin. The eflect of retinoids on expression of other
glycosyltransferases is at present unknown.
In summary, we detnonstrate that the glycosylation
pattern of SLS-treated epidermis is not significantly
different from that seen in normal skin and that RAtreated epidermis strongly expresses Le^ and minimally
expresses T antigen—features of mucosal epithelia.
These observations are compatible with the idea that
topical RA and irritants have differing effects on the
epidermis.
Acknowledgments
This work was supported in part by the R.WJohnson
Pharmaceutical Research Institute. Raritan, NJ, U.S.A.,
the Babcock Dermatoiogic Endowment Fund. Ann
Arbor. ML U.S.A.. and the Meta and Hakon Baggers
Foundation. Denmark. We are grateful to Mr Ted
A.Hamilton for statistical analysis.
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