Vol. 22, No. 3, 2010
Received February 2, 2010, Revised March 8, 2010, Accepted for
publication March 8, 2010
*This study was supported by a grant from the SKINMED
CORPORATION. This study was also supported by a grant from the
National Research Foundation of Korea (KRF-2008-314-E00152).
Corresponding author: Jeung-Hoon Lee, M.D., Department of Derma-
tology, School of Medicine, Chungnam National University, 33
Munhwa-ro, Daejeon 301-040, Korea. Tel: 82-42-280-7707, Fax:
82-42-280-8459, E-mail: email@example.com
Ann Dermatol Vol. 22, No. 3, 2010DOI: 10.5021/ad.2010.22.3.255
Enhancement of Keratinocyte Differentiation by Rose
Jin-Hwa Kim, Ph.D., Dae-Kyoung Choi, Ph.D.1, Sang-Sin Lee, M.D.1, Sun Ja Choi, Ph.D.2,3,
Chang Deok Kim, Ph.D.1, Tae-Jin Yoon, M.D., Jeung-Hoon Lee, M.D.1
Department of Dermatology and Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju, 1Department of
Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, 2Department of Visual
Design, Daeduk University, Daejeon, 3Department of Advanced Organic Materials Engineering, Chonbuk National University, Jeonju,
Background: Through differentiation processes, keratino-
cytes provide a physical barrier to our bodies and control skin
features such as moisturization, wrinkles and pigmentation.
Keratinocyte differentiation is disturbed in several skin
diseases such as psoriasis and atopic dermatitis. Objective:
The aim of this study is to evaluate the keratinocyte
differentiation-enhancing effect of rose absolute oil (RAO).
Methods: Primary cultured human normal keratinocytes
were treated with RAO, and differentiation then checked by
the expression of marker genes. Results: RAO did not induce
cytotoxicity on cultured keratinocytes at a dose of 10μM.
The level of involucrin, an early marker for keratinocyte
differentiation, was significantly increased by RAO. Con-
comitantly, RAO increased involucrin promoter activity,
indicating that RAO increased involucrin gene expression at
the mRNA level. Furthermore, RAO increased the level of
filaggrin in cultured keratinocytes, and in the granular layer
of mouse skin. In line with these results, RAO decreased the
proliferation of keratinocytes cultured in vitro. When RAO
was applied topically on the tape-stripped mouse skins, it
accelerated the recovery of disturbed barrier function.
Conclusion: These results suggest that RAO may be
applicable for the control of skin texture and keratinocyte
differentiation-related skin diseases. (Ann Dermatol 22(3)
Differentiation, Filaggrin, Keratinocyte, Rose absolute oil
In the epidermis, keratinocytes bear most responsibility for
maintaining structure and homeostasis. Epidermal kera-
tinocytes provide the rigid stratified structure through a
sophisticated differentiation program1,2. Keratinocyte di-
fferentiation involves the process of cell cycle arrest and
the onset of expression of numerous genes, resulting in,
characteristically, 4 layers of epidermis (stratum basale,
stratum spinosum, stratum granulosum and stratum
corneum)3,4. The transition from basal cells to corneocytes
is a complex process that requires the simultaneous
activation and inactivation of a wide variety of genes5. It
has been established that many genes such as involucrin,
loricrin and filaggrin are expressed in a temporally regul-
ated manner during keratinocyte differentiation6.
Dysregulated keratinocyte differentiation is also closely
related with several skin diseases including psoriasis and
atopic dermatitis7-9. Interestingly, such complex inflamma-
tory skin diseases are associated with hyperproliferation of
keratinocytes and disruption of skin barrier function,
resulting in exacerbation of immunologic reaction and
inflammation. Concordantly, disruption of skin barrier
function leads to excessive dry skin, which may be
another exacerbating factor for differentiation-related skin
diseases10. To date, major modalities for these skin dis-
eases are linked to the inactivation of immune reactions,
JH Kim, et al
such as cyclosporine A, tacrolimus and pimecrolimus11.
Additionally, much evidence indicates that moisturization
of skin has a beneficial effect on reducing disease status
and enhancing skin texture12. It is known that the final
products of keratinocyte differentiation, such as filaggrin,
provide natural moisturizing properties, thereby allowing
for the maintenance of healthy skin13-15. Thus, we can
envisage the usage of therapeutic agents which enhance
keratinocyte differentiation in conjunction with first-line
treatment agents such as immunosuppressives. In this
study, using an in vitro culture system, we demonstrate
that rose absolute oil (RAO) can enhance keratinocyte
differentiation, suggesting that RAO can be used to
strengthen skin texture.
MATERIALS AND METHODS
Normal human skin samples were obtained from
circumcisions, in accordance with a process approved by
the ethical committee of Chungnam National University
Hospital. Keratinocytes were primary cultured as pre-
viously reported16. Briefly, specimens were sterilized in
70% ethanol, minced, and then treated with dispase
overnight at 4oC. The epidermis was separated and placed
in a solution containing 0.05% trypsin and 0.025%
ethylenediaminetetraacetic acid (EDTA) at 37oC for 15
min. After vigorous pipetting, cells were pelleted and
resuspended in keratinocyte-serum free medium (K-SFM)
supplemented with bovine pituitary extract and recom-
binant human epidermal growth factor (Invitrogen, Grand
Island, NY, USA).
Keratinocytes (2×105) were seeded on 12-well culture
plates and incubated overnight. After treatment with RAO,
cells were replenished with fresh medium. After incuba-
tion for the indicated time points, cells received 2 mg/ml
MTT solution and were incubated for a further 4 h. The
medium was removed and the resulting formazan crystal
was solubilized in 100μl of dimethylsulfoxide (DMSO).
The optical density at 540 nm was determined using an
enzyme-linked immunosorbent assay (ELISA) reader.
Western blot analysis
Cells were lysed in Proprep solution (Intron, Daejeon,
Korea). Total protein was measured using a Bradford
protein assay kit (Bio-Rad Laboratories, Hercules, CA,
USA). Samples were run on sodium dodecyl sulfate
(SDS)-polyacrylamide gels, transferred onto nitrocellulose
membranes and incubated with appropriate antibodies.
Blots were then incubated with peroxidase-conjugated
secondary antibodies, visualized by enhanced chemilumi-
nescence (Intron, Daejeon, Korea). The following primary
antibodies were used in this study: involucrin (Santa Cruz
Biotechnologies, Santa Cruz, CA, USA), filaggrin (Co-
vance, Princeton, NJ, USA), and actin (Sigma, St. Louis,
Creation of recombinant adenovirus
For creation of involucrin-luc and loricrin-luc reporter
adenoviruses, genomic DNA isolated from keratinocytes
was used as a template for polymerase chain reaction
(PCR). Primer sequences were as follows: involucrin
promoter, 5’-CTCCATGTGTCATGGGATATG and 5’-TCA-
ACCTGAAAGACAGAAGAG. The amplified DNA frag-
ment was subcloned into pENT/GL3 vector that contained
attL sites for site-specific recombination with a Gateway
destination vector. The replication-incompetent adeno-
viruses were created using Virapower adenovirus ex-
pression system (Invitrogen, Grand Island, NY, USA)
according to the method previously described17. Briefly,
site-specific recombination between entry vector and
adenoviral destination vector was achieved by LR clonase.
The resulting adenoviral expression vector was then
transfected into 293A cells using Lipofectamine 2000
(Invitrogen, Grand Island, NY, USA). Cells were grown
until 80% cytopathic effect (CPE) was seen, then harvested
for preparation of recombinant adenovirus.
Keratinocytes were grown at 50% confluency in 12-well
culture plates, transduced with reporter adenovirus. After
adenoviral transduction, cells were replenished with fresh
medium and treated with RAO. Cells were further
incubated for 48 h, and then cellular extracts were
prepared using cell lysis buffer. Luciferase activities were
determined using Luciferase assay system (Promega,
Madison, WI, USA), according to the recommended
Paraffin sections of skin specimens were de-waxed,
re-hydrated and washed 3 times with phosphate buffered
saline (PBS). Sections were then incubated with proteinase
K (Dako, Carpinteria, CA, USA) for 5 min at 37oC, and
treated with H2O2 for 10 min at room temperature,
blocked in 0.1% Tween-20, 1% bovine serum albumin in
PBS for 30 min, and followed by reaction with anti-
filaggrin antibody (Abcam, Cambridge, MA, USA) for 1 h.
Sections were incubated sequentially with peroxidase-
conjugated secondary antibody and visualized with Che-
Keratinocyte Differentiation by Rose Absolute Oil
Vol. 22, No. 3, 2010
Fig. 1. Cytotoxicity of rose absolute oil (RAO). Normal human
epidermal keratinocytes were treated with RAO at the indicated
concentrations for 3 days. Cell viability was measured by MTT
assay. The mean values±SEM are averages of triplicate
Fig. 2. Effect of rose absolute oil (RAO) on involucrin expression in keratinocytes. (A) Normal human epidermal keratinocytes were
treated with RAO at the indicated concentrations for 3 days. Calcium (1.2 mM) was included as a positive control for keratinocyte
differentiation. Cellular proteins were prepared and protein level for involucrin was evaluated by Western blot. Actin was used as
a loading control. (B) Effect of RAO on the involucrin promoter activity. Keratinocytes were transduced with 10 multiplicity of infections
(MOIs) of involucrin-luc reporter adenovirus, then treated with RAO for 3 days. Cells were lysed and assayed for luciferase activity.
Data are represented as relative light unit (RLU) and SEM, measured from 3 independent experiments.
mmate envision detection kit (Dako, Carpinteria, CA,
USA). Sections without primary antibody were used as
Cell growth analysis
For [3H]thymidine uptake assay, keratinocytes were
seeded in a 60-mm culture dish and treated with 1μCi of
[3H]thymidine (Amersham, Buckinghamshire, UK). Fol-
lowing incubation for the indicated time point, cells were
washed twice with PBS and incubated with 0.1 N NaOH
at room temperature. Radioactivity in cell lysates was
measured by liquid scintillation counter.
Transepidermal water loss
Seven-week-old female hairless mice were purchased
from Orient Bio Inc. (Seongnam, Korea). Skin barrier was
disturbed by tape-stripping, then RAO was topically
applied. RAO was dissolved in 70% polyethylene glycol
and 30% ethanol. Transepidermal water loss (TEWL) was
measured using an evaporimeter (Tewameter TM210,
Courage＋Khazaka, Koln, Germany), as previously des-
To investigate the cytotoxicity of RAO on keratinocytes,
we treated normal human epidermal keratinocytes with
RAO serially. After treatment for 3 days, cell viability was
checked by MTT assay. As shown in Fig. 1, RAO showed
no cytotoxicity up to a dose of 10μg/ml. Next, we
determined the effect of RAO on keratinocyte differentia-
tion by examining the protein level of involucrin, an early
marker for differentiation. Calcium, the best-known differ-
entiation inducer, was included as a positive control. After
treatment for 3 days, the protein level for involucrin was
markedly increased by RAO in a dose-dependent manner
(Fig. 2A). To investigate whether RAO affected the
involucrin expression at the promoter level, we tested the
involucrin promoter activity using a recombinant adeno-
virus harboring reporter construct in which sequences 3.0
kb upstream of the translation start site of the involucrin
JH Kim, et al
Fig. 3. Effect of rose absolute oil (RAO) on filaggrin expression.
Normal human epidermal keratinocytes were treated with RAO
at the indicated concentrations for 7 days. Calcium (1.2 mM)
was included as a positive control for keratinocyte differentiation.
Cellular proteins were prepared and protein level for filaggrin
was evaluated by Western blot. Actin was used as a loading
Fig. 4. Effect of rose absolute oil (RAO) on the cell growth.
Normal human epidermal keratinocytes were treated with 10μg/ml
RAO for the indicated time points in the presence of 1μCi of
[3H]thymidine. Calcium (1.2 mM) was included as a positive
control for keratinocyte differentiation. Cells were lysed using
0.1 N NaOH and radioactivity was measured. Data are expressed
as percentage of control. The mean values±SEM are averages
of triplicate measurements.
gene were fused to the luciferase gene. As expected, RAO
induced luciferase activity in a dose-dependent manner,
similar to that of calcium treatment (Fig. 2B). Filaggrin, the
late differentiation marker for keratinocyte differentiation,
was also checked by Western blotting, after long-term
treatment with RAO. Interestingly, RAO treatment resulted
in significant induction of filaggrin after 7 day treatment
(Fig. 3). To further investigate the effect of RAO in vivo,
we topically applied RAO on mouse skin. After 7 day
treatment, the expression of filaggrin was evaluated by
immunohistochemistry. RAO treatment led to a significant
increase of filaggrin in the granular layer of mouse skin,
potentiating its keratinocyte differentiation-enhancing
As the differentiation process takes place along a pathway
that leads to concomitant cell cycle arrest, we next
evaluated the effect of RAO on cell growth using
[3H]thymidine uptake assay. As anticipated, RAO treat-
ment led to the retardation of cell growth, consistent with
the results of enhancing keratinocyte differentiation (Fig. 4).
TEWL is a well established indicator that reveals the
disturbance of skin barrier function. To investigate the
effect of RAO on skin barrier function, we disturbed
mouse skin barrier with a tape-stripping method. When
topically applied, significant acceleration of barrier
recovery was observed after 4 h treatment of RAO. To
enhance the keratinocyte differentiation further, we also
formulated a RAO-containing complex in which RAO
(0.1%), oleic acid (0.1%), oleanolic acid (0.05%),
urocanic acid (0.01%), acetylhexapeptide (0.01%) and
ceramide (0.1%) were additionally included. Interestingly,
this RAO complex also markedly accelerated the barrier
recovery (Fig. 5A). In line with these results, immuno-
histochemistry showed that filaggrin expression was
significantly increased in RAO- and RAO complex-treated
groups (Fig. 5B).
In epidermis, basal layer keratinocytes proliferate and
move upwards, with the differentiation process beginning
in the suprabasal layer and culminating in fully differ-
entiated dead cells on the surface19. The resultant corni-
fied layers function as a physical barrier to protect the
organism from the environment. It is well known that
much of this barrier function is provided by the cornified
cell envelope (CE), a specialized insoluble structure
formed beneath the plasma membrane in terminally
differentiated stratified squamous epithelium20. The CE is
built by the cross-linking of various components including
filaggrin, involucrin, loricrin and small proline-rich (SPR)
proteins in the plasma membrane21,22. Among them,
filaggrin aggregates with keratins to form macrofilaments
after cleavage of polymeric proteins into monomers.
Filaggrin monomers are also degraded into small amino
acid molecules, which function as natural moisturizing
factors in the stratum corneum and as UV radiation
filters23. Interestingly, it has been shown that wrinkle
formation is closely related with thinning of epidermis
together with filaggrin reduction24. It has also been
suggested that stress to the skin barrier promotes the
pigmentation process25. Furthermore, filaggrin expression
is elevated in reepithelialized epithelium during human
Keratinocyte Differentiation by Rose Absolute Oil
Vol. 22, No. 3, 2010
Fig. 5. Effect of rose absolute oil (RAO) on transepidermal water loss (TEWL). (A) Hairless mice were tape-stripped, then topically
treated with 0.1% solution of RAO (in 70% polyethylene glycol and 30% ethanol) and RAO complex in which RAO (0.1%), oleic
acid (0.1%), oleanolic acid (0.05%), urocanic acid (0.01%), acetylhexapeptide (0.01%) and ceramide (0.1%) were additionally included.
At the indicated time points, TEWL was measured using Tewameter. Basal TEWL (0 h) was measured before tape-stripping. Five
hairless mice were included in each group. Data are represented as average and SEM. (B) Skin specimens were obtained after 5
days of treatment. Sections were prepared and stained with anti-filaggrin antibody (×100).
cutaneous wound healing26. In this regard, it could be
suggested that strengthening of skin texture by enhancing
keratinocyte differentiation may also influence wrinkle
formation, pigmentation and skin regeneration.
Many studies indicate that disruption of the cutaneous
barrier is closely associated with skin disorders such as
psoriasis and atopic dermatitis. But controversy still
remains as to whether the cause of differentiation-related
skin diseases is primarily dependent on perturbation of
skin barrier or the onset of abnormal inflammatory
immune reaction27. Despite the debatable evidence,
however, it is generally accepted that skin manifestations
are directly linked to the dysregulated keratinocyte
differentiation, including epidermal hyperproliferation,
changes in keratin compositions, and down-modulation of
the cornified envelope protein filaggrin28,29. Therefore, an
approach that enhances keratinocyte differentiation,
thereby normalizing the skin texture, is an attractive
method for curing differentiation-related skin diseases. In
this study, we attempted to validate the keratinocyte
differentiation-enhancing effect of a candidate agent using
primary cultured keratinocytes. We demonstrated that
RAO is able to enhance keratinocyte differentiation and
inhibit cell proliferation. In addition, RAO upregulated
filaggrin production and accelerated the recovery of
disturbed skin barrier, suggesting that it is able to exert
beneficial effects on skin texture through the increase of
natural moisturizing factors.
The main component of RAO is phenethyl alcohol
(C6H5CH2CH2OH), a phenolic compound. The phenethyl
alcohol can be obtained from a variety of plant sources
including rose, carnation, hyacinth and orange blossom. It
has been demonstrated that this phenolic compound has
antioxidant and antibacterial effects30. Interestingly,
several studies indicate that antioxidants have profound
effects on keratinocyte differentiation. For example,
N-acetyl L-cysteine (NAC) induces a 10-fold more rapid
differentiation in normal primary keratinocytes, arrests the
cell cycle and promotes cytoskeletal reorganization31.
Other evidence indicates that the antioxidant epigallo-
catechin gallate (EGCG) promotes keratinocyte differen-
tiation32. In this context, we speculate that the kerati-
JH Kim, et al
nocyte differentiation-promoting effect of RAO may be
related to its antioxidant potential. However, the precise
mechanism by which phenethyl alcohol effects keratino-
cyte differentiation remains to be elucidated.
In our study, RAO treatment resulted in increase of
involucrin promoter activity. Since phenolic antioxidant
triggers activation of mitogen-activated protein kinase
(MAPK) signaling and induction of phase II/III drug
metabolizing enzymes, it may be that RAO influences the
intracellular signaling cascades related with keratinocyte
differentiation. Elucidation of precise intracellular signal-
ing induced by RAO will be an interesting topic for further
In conclusion, RAO has the potential to enhance kera-
tinocyte differentiation and inhibit cell proliferation. Our
results suggest that RAO may be applicable for the control
of skin texture and keratinocyte differentiation-related skin
diseases in conjunction with first-line treatments.
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