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230

Journal of Pharmacological Sciences


The Japanese Pharmacological Society
J Pharmacol Sci 115, 230 234 (2011)
Pantothenic acid is one of the essential nutrients con-
tained in various foods. Pantothenic acid, also referred to
as vitamin B5, is a member of the water-soluble group of
vitamins. Pantothenic acid is converted to 4-phospho-
pantetein, which is then converted to coenzyme A (CoA)
with ATP. CoA is involved in acyl transfer during me-
tabolism of carbohydrates, amino acids, and lipids. CoA
is also utilized for synthesis of acyl carrier protein (ACP),
which is mainly involved in lipid metabolism. Although
pantothenic acid deficiency is rare in healthy subjects,
factors such as stress, pregnancy, and predominant con-
sumption of processed foods can cause pantothenic acid
deficiency. It has been reported that pantothenic acid
deficiency causes growth arrest, weight loss, dermatitis,
dyslipidemia, neuropathy, and adrenal disorders (1, 2).
Pantothenic acid and its alcohol analogue panthenol
have been used for many years for skin protection and to
improve skin regeneration after wounding (3). In terms
of the effects of pantothenic acid on dermal fibroblasts,
Weimann and Hermann (4) revealed that Ca D-panto-
thenic acid promoted fibroblast proliferation and migra-
tion. Furthermore, Wiederholt et al. (5) used microarray
analysis to investigate the effects of Ca D-pantothenic
acid on gene expression in proliferating dermal fibro-
blasts. However, there are no studies examining the effect
of pantothenic acid on keratinocytes. Therefore, in this
study, we examined the direct effect of pantothenic acid
on the proliferation and differentiation of keratinocytes
by using the MTT assay and measuring the expression of
cytokeratin 10 (CK10), a keratinocyte differentiation
marker (6). We also evaluated the effect of pantothenic
acid deficiency on the synthesis of keratinocyte growth
factor (KGF) and procollagen in fibroblasts, which are
involved in keratinocyte growth.
Human HaCaT keratinocytes were given to us by Dr.
N.E. Fusenig (German Cancer Research Center,
Heidelberg, Germany). The cells were grown in Dul-
beccos modified Eagles medium (DMEM) containing
4 mg/l Ca D-pantothenic acid supplemented with 1% fetal
calf serum (FCS), penicillin (50 units/ml), and strepto-
mycin (50 g/ml) in a humidified atmosphere of 5%
CO
2
/ 95% air at 37C. Keratinocytes were treated with
pantothenic aciddeficient DMEM for a week before the
experiments. Fibroblasts were prepared as follows: Em-
bryonic day 15.5 mice were dissected and the whole
body except head and extremities were homogenized,
*Corresponding author. nakahata@mail.pharm.tohoku.ac.jp
Published online in J-STAGE on January 18, 2011 (in advance)
doi: 10.1254/jphs.10224SC
The Effect of Pantothenic Acid Deficiency on Keratinocyte Proliferation
and the Synthesis of Keratinocyte Growth Factor and Collagen
in Fibroblasts
Daisaku Kobayashi
1
, Miho Kusama
1
, Masaaki Onda
2
, and Norimichi Nakahata
1,
*
1
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University,
6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
2
Daiichi Fine Chemical Co., Ltd., 530 Chokeiji, Takaoka, Toyama 933-8511, Japan
Received August 27, 2010; Accepted December 3, 2010
Abstract. It has been reported that pantothenic acid (vitamin B5) and panthenol, an alcohol de-
rivative of pantothenic acid, have beneficial moisturizing effects on the skin. However, few studies
have investigated the mechanism of action of pantothenic acid on skin tissues. We tried to clarify
the role of pantothenic acid on skin function by using keratinocytes and fibroblasts. The depletion
of pantothenic acid from the culture medium suppressed keratinocyte proliferation and promoted
differentiation. Moreover, pantothenic acid depletion decreased the synthesis of keratinocyte
growth factor and procollagen 4a2 in fibroblasts. These results suggest that pantothenic acid is
essential for maintaining keratinocyte proliferation and differentiation.
Keywords: collagen, keratinocyte, pantothenic acid
Short Communication
231 Pantothenic Acid Regulates Skin Functions
washed with phosphate-buffered saline (PBS) many
times. They were cultured throughout in normal DMEM
or pantothenic aciddeficient DMEM supplemented with
10% FCS, penicillin (50 units/ml), and streptomycin (50
g/ml) in a humidified atmosphere of 5% CO
2
/ 95% air
at 37C for a week and used for the experiments. Cell
viability was measured by the previously described MTT
assay (7). The extraction of total RNA and the prepara-
tion of first-strand cDNA was done as previously de-
scribed (8). The real-time PCR conditions were as fol-
lows: 94C for 10 s, followed by several cycles of 10 s at
94C, 20 s at the annealing temperature shown in Table
1, and 30 s at 72C. The mRNA expressions of CK10,
KGF, and procollagen 1a1, 1a2, 4a1, and 4a2 were each
normalized by the expression of -actin. The protein
synthesis of CK10 and procollagen 4a2 were analyzed by
SDS-polyacrylamide gel electrophoresis and immunob-
lotting. HaCaT cells and mouse fibroblasts were dis-
solved in Laemmli sample buffer (final concentration: 75
mM Tris-HCl, 2% SDS, 15% glycerol, and 3% 2-mer-
captoethanol, pH 6.8) and heated at 95C for 5 min.
Electrophoresis was performed on 10% or 8% acrylamide
gels. Proteins were transferred electrically from the gel
onto a polyvinylidene difluoride membrane (Millipore
Corporation, Billerica, MA, USA) by a semidry blotting
method. The blots were blocked for 1 h with 5% skim
milk in tris-buffered saline supplemented with 0.1%
Tween 20, and then they were incubated with a 1:1000
dilution mouse anti-CK10 antibody (Thermo Fisher
Scientific, Inc., Waltham, MA, USA) or rabbit anti-pro-
collagen 4a2 antibody (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA, USA) overnight at 4C. After several
washes, the blots were incubated at 25C for 1 h with a
1:4000 dilution of secondary antibody (horseradish per-
oxidaseconjugated anti-rabbit IgG antibody or anti-
mouse IgG antibody). Blots were developed using an
enhanced chemiluminescence on Hyperfilm. Data are
expressed as means S.E.M. Significant differences
were determined by Students t-test or Mann-Whitneys
U test.
It has been shown that pantothenic acid affects cell
proliferation of dermal fibroblasts (4). However, there
are no studies investigating the effect of pantothenic acid
on keratinocyte proliferation. Therefore, we first investi-
gated the effect of pantothenic acid deficiency on the
proliferation of HaCaT keratinocytes using the MTT as-
say (Fig. 1A) and cell counting (Fig. 1B). The MTT assay
and cell counting revealed that the proliferation of HaCaT
keratinocytes cultured in pantothenic aciddeficient
medium was suppressed compared with HaCaT keratino-
cytes cultured in the control medium. Then, we examined
the effect of pantothenic aciddeficient medium on the
differentiation of keratinocytes, determining the express-
tion of CK10, a keratinocyte differentiation marker (6).
The mRNA expression of CK10 (Fig. 1C) and the protein
level of CK10 (Fig. 1D) were promoted in pantothenic
aciddeficient medium. These data suggest that the dif-
ferentiation of HaCaT keratinocytes cultured in panto-
thenic aciddeficient medium was enhanced compared
with HaCaT keratinocytes cultured in the control medium.
The low proliferative ability and enhanced differentiation
of keratinocytes cultured in pantothenic aciddeficient
DMEM indicate that the depletion of pantothenic acid
may decelerate or arrest the cell cycle, thus enhancing
keratinocyte differentiation (9). It is known that the bal-
ance between proliferation and differentiation is regulated
Table 1. Primers for reverse transcription-polymerase chain reaction used in the present study
Sequence Accession number
Annealing
temperature (C)
CK10
KGF
Procollagen 1a1
Procollagen 1a2
Procollagen 4a1
Procollagen 4a2
-Actin
Sense:
Anti-sense:
Sense:
Anti-sense:
Sense:
Anti-sense:
Sense:
Anti-sense:
Sense:
Anti-sense:
Sense:
Anti-sense:
Sense:
Anti-sense:
5-GGATGAGCTGACCCTGACCAA-3
5-GCAGCATTCATTTCCACATTCAC-3
5-TTTGGAAAGAGCGACGACTT-3
5-GGCAGGATCCGTGTCAGTAT-3
5-GCAACATGGAGACAGGTCAG-3
5-TCGCTTCCGTACTCGAACG-3
5-CCGTTGGCAAAGATGGTAG-3
5-CGTTGTCGTAGCAGGGTTCT-3
5-TTCGTTGGCCTCTGTTTGC-3
5-TTGGGGCTGAAACGATGG-3
5-CCAGCACTCATTCCAACC-3
5-GACTGCAGGAAGGTGAGC-3
5-AGGGAAATCGTGCGTGACAT-3
5-TCCTGCTTGCTGATCCACAT-3
NM_000421.3
NM_008008.4
NM_007742.3
NM_007743.2
NM_009931.2
NM_009932.3
NM_03114
60
56
56
56
56
56
56
232 D Kobayashi et al
by various factors, and protein kinase C or tyrosine kinase
may be involved in the switch from proliferation to
differentiation (10), although the detailed mechanism has
not been reported. Moreover, it has been shown that an
increased intracellular Ca
2+
concentration or high cell
density might induce cell differentiation (11). Our data
suggest that pantothenic aciddeficiency might induce
keratinocyte differentiation directly, accompanied with
growth arrest, although the mechanism of the pantothenic
acid is still unknown.
Fibroblasts secrete various kinds of growth factors and
active substrates, which regulate keratinocyte prolifera-
tion and differentiation (12). Then, we examined the
synthesis of KGF, also referred to FGF7, and procollagen
in fibroblasts cultured in pantothenic aciddeficient
medium.
It is known that KGF is secreted by fibroblasts and
promotes keratinocyte proliferation and migration (12).
Our results indicated that the mRNA expression of KGF
was decreased in pantothenic aciddeficient fibroblasts
(Fig. 2A). Since the suppression of the KGF level causes
the growth arrest of keratinocytes, it is possible that
pantothenic acid promotes the growth of keratinocytes
via the synthesis of KGF.
Collagen is categorized into more than twenty func-
tional types, including fibrous collagen (type I, II, III, V,
XI), basement membrane collagen (type IV), long-chain
collagen (type VII), and short-chain collagen (VIII, X).
All types of collagen are important for the regulation of
the function of epidermal cells (13). Next we examined
the synthesis of collagens in fibloblasts. First, we focused
on type I collagen and evaluated procollagen 1a1 and 1a2
mRNA expression. Procollagen is a precursor of mature
collagen, and type I collagen consists of two procollagen
1a1 and one procollagen 1a2 fibers. We found that there
was no difference in type I procollagen mRNA expres-
sion between the normal medium and the pantothenic
aciddeficient medium (Fig. 2: B, C). Next, the expres-
sion of type IV basement membrane procollagen was
examined. We found that the mRNA expression of pro-
collagen 4a1 and 4a2 was partially suppressed in fibro-
blasts cultured in the pantothenic aciddeficient medium
(Fig 2: D, E). Furthermore, the protein level of procol-
lagen 4a2 was also decreased in fibroblasts cultured in
the pantothenic aciddeficient medium (Fig. 2F). Thus,
pantothenic acid may be involved in the biosynthesis of
type IV collagen, which is important for the adherence
and growth of epidermal cells (14).
(A)
Control PA (-)
A
b
s
o
r
b
a
n
c
e

(
5
9
5

n
m
)
0
0.5
1.0
1.5
2.0
*
Control PA (-)
0
0.2
0.4
0.6
0.8
1.0
1.2
C
e
l
l

n
u
m
b
e
r
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
*
(B)
(C)
Control PA (-)
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
*
C
K
1
0

m
R
N
A

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
(D)
Control
-Actin
Cytokeratin 10
PA (-)
Fig. 1. Effect of pantothenic acid deficiency on the proliferation of HaCaT keratinocytes. Keratinocytes were cultured in normal
DMEM or pantothenic aciddeficient DMEM. A) Cell growth was measured by the MTT assay. Data are expressed as
means S.E.M. of three determinations. *P < 0.05 vs. control. B) The cells were detached and the cell number was counted. Data
are expressed as means S.E.M. of 11 determinations. *P < 0.05 vs. control. C) CK10 mRNA expression was analyzed by RT-
PCR. Data are expressed as means S.E.M. of six determinations. *P < 0.05 vs. control. D) CK10 protein expression was detected
by Western blotting.
233 Pantothenic Acid Regulates Skin Functions
Keratinocyte growth and differentiation are essential
for epidermal barrier function (15). The present results
suggest that pantothenic acid may regulate epidermal
barrier function through the proliferation and differentia-
tion of keratinocytes directly or indirectly via the synthe-
sis of KGF and type IV collagen.
Acknowledgments
This work was partly supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology (No, 19659011 and 20054002 to N.N.) and by a grant
from the Smoking Research Foundation (N.N.). This work was also
partly supported by the Sasakawa Scientific Research Grant from The
Japan Science Society (D.K.).
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234 D Kobayashi et al
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