Download Malva verticillata seed extracts upregulate the Wnt pathway in

International Journal of Cosmetic Science, 2016, 38, 148–154
doi: 10.1111/ics.12268
Malva verticillata seed extracts upregulate the Wnt pathway in
human dermal papilla cells
E. Y. Lee*,a, E.-J. Choi*,a, J. A. Kim†, Y. L. Hwang‡, C.-D. Kim§, M. H. Lee‡, S. S. Roh‡, Y. H. Kim¶, I. Han** and S. Kang*
*Department of Biotechnology, CHA University, Seongnam, Korea, †College of Pharmacy, Kyungpook National University, Daegu, Korea, ‡OBM
Laboratory, Daejeon, Korea, §Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, Korea, ¶College of Pharmacy,
Chungnam National University, Daejeon, Korea and **Department of Neurosurgery, CHA University, CHA Bundang Medical Center, Seongnam,
Korea
Received 18 June 2015, Accepted 28 July 2015
Keywords: dermal papilla cells, hair growth, Malva verticillata seeds, myristoleic acid, Wnt/b-catenin
Synopsis
OBJECTIVE: Mesenchymal–epithelial interactions are important in
controlling hair growth and the hair cycle. The b-catenin pathway
of dermal papilla cells (DPCs) plays a pivotal role in morphogenesis
and normal regeneration of hair follicles. Deletion of b-catenin in
the dermal papilla reduces proliferation of the hair follicle progenitor cells that generate the hair shaft and induces an early onset of
the catagen phase. In this study, a modulator of the Wnt/b-catenin
activity was studied in oriental herb extracts on cultured human
DPCs.
METHODS: The effect of Malva verticillata (M. verticillata) seeds on
human DPCs was investigated by a Wnt/b-catenin reporter activity
assay system (b-catenin–TCF/LEF reporter gene) and cell proliferation analysis. The synthesis of the factors related to hair growth
and cycling was measured at both the mRNA and the protein level
by semi-quantitative PCR and Western blot analysis, respectively.
RESULTS: An extract from M. verticillata seeds increased Wnt
reporter activity in a concentration-dependent manner and also led
to increased b-catenin levels in cultured human DPCs. Myristoleic
acid, identified as an effective compound of M. verticillata seeds,
stimulated the proliferation of DPCs in a dose-dependent manner
and increased transcription levels of the downstream targets: IGF1, KGF, VEGF and HGF. Myristoleic acid also enhanced the phosphorylation of MAPKs (Akt and p38).
CONCLUSION: Overall, the data suggest that this extract of
M. verticillata seeds could be a good candidate for treating hair loss
by modulating the Wnt/b-catenin pathway in DPCs.
sume
Re
OBJECTIF: Les interactions mesenchymateuses- epitheliales sont
importantes dans le contr^
ole de la croissance des cheveux et du
cycle pilaire. La voie b-catenine des cellules des papilles dermiques
(CPD) joue un r^
ole essentiel dans la morphogenese et la regeneration normale des follicules pileux. La suppression de la b-catenine
dans la papille dermique reduit la proliferation des cellules souches
du follicule pileux qui generent la tige du cheveu et induit une
apparition precoce de la phase catagene. Dans cette etude, un modCorrespondence: Sangjin Kang, Department of Biotechnology, CHA
University, Seongnam, Korea. Tel./fax: +82-31-881-7020;
e-mail: [email protected]
a
These two authors contributed equally to this work.
148
ulateur de l’activite/b-catenine Wnt a ete etudiee dans des extraits
d’herbes orientales sur CPD humains cultives.
METHODES:
L’effet des graines de mauve crepue (Malva verticillata) sur les CPD humaines a ete etudie par un systeme de dosage
de l’activite du gene rapporteur Wnt/b-catenine (TCF-b-catenine/
gene rapporteur LEF) et l’analyse de la proliferation cellulaire. La
la croissance des cheveux et au cycle a
synthese des facteurs lies a
ete mesuree a
la fois au niveau d’ARNm et des proteines par PCR
semi-quantitative et une analyse par Western blot, respectivement.
RESULTATS:
Un extrait des graines de M. verticillata a augmente
l’activite de Wnt rapporteur d’une maniere dependante de la con des niveaux accrus de bcentration et a egalement conduit a
catenine dans les CPD humaines en culture. L’acide myristoleique,
identifie comme etant un compose efficace des graines de M. verticillata, a stimule la proliferation des CPD d’une maniere dependante de
la dose ainsi que l’augmentation des niveaux des cibles en aval de
l’IGF-1, KGF, VEGF, HGF et leur transcription. L’acide myristole€ıque
a egalement ameliore la phosphorylation des MAPK (Akt et p38).
CONCLUSION: Globalement, les donnees suggerent que cet extrait
des semences de M. verticillata pourrait ^etre un bon candidat pour
le traitement de la perte de cheveux en modulant la voie/bcatenine dans les CPD.
Introduction
Hair follicles are a unique organ that regenerate themselves during
the normal human lifespan [1]. Hair follicles undergo a repetitive
regeneration process in the ‘hair cycle’ which consists of growth
(anagen), regression (catagen) and rest (telogen) phases [2]. During
the anagen phase, the pigmented hair shaft is actively generated
and the follicle reaches its maximal length and volume. Once the
anagen phase is completed, the hair follicle enters the catagen
phase; production of new hair shafts and pigmentation ceases and
the club hair forms. The telogen phase is a relatively quiescent
state during which keratin production stops and the club hair
matures. When the telogen phase is completed, the hair is shed
and the cycle begins again [3].
Hair follicles consist of epithelial and mesenchymal cells, and
interactions between these two cell types play pivotal roles in follicle morphogenesis and hair growth. The dermal papilla, a mesenchymal cell population located at the base of the hair follicle,
plays an important role in regulating hair growth and cycling [4].
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
M. verticillata seed extracts upregulate the Wnt pathway
E. Y. Lee et al.
Seeds of Malva verticillata
EtOH extract
59A
Methylene chloride soluble fraction
59B
H2O soluble fraction
59D
EtOAc soluble fraction
59C
Silica gel C.C. n-Hex/EtOAc/MeOH/H2O
Figure 1 Isolation of compounds from Malva
verticillata seeds. Six compounds (60B, 61E,
59E, 62B, 63B and 63C) were isolated from the
methylene chloride extract of M. verticillata
seeds using a combination of silica gel and
reverse C18 column chromatography. These
compounds were identified by comparison of
their physical and spectral data with literature
values as b-sitosterol (60B), verticilloside (61E),
daucosterol (59E), sucrose (62B), raffinose
(63C) and myristoleic acid (63B).
59B-1~3
59B-4
60A
59B-5~6:
61A
Silica gel C.C. Silica gel C.C.
n-Hex/EtOAc CH2Cl3/MeOH
60B
Factors secreted from dermal papilla cells (DPCs) directly influence
the surrounding matrix cells either to proliferate and differentiate
or to stimulate hair stem cells to initiate a new anagen phase [5].
The Wnt/b-catenin pathway is known to be essential for these biological processes in hair follicles [6–10]. Recently, it was reported
that deletion of Wnt signalling reduced the proliferation of hair follicle progenitor cells and induced early onset of the catagen phase
[11]. Upregulation of Wnt signalling has resulted in more extensive
hair growth in mice [7].
Hair loss is associated with alterations of the hair cycle such as
decreased growth rate, an early transition from the anagen to catagen phase and a delay or absence of hair regeneration [12]. Minoxidil and finasteride are the only treatments approved by the FDA
for the treatment of female pattern hair loss and androgenic alopecia [13, 14]; however, these medications are associated with significant side effects and are the basis of therapy for men. For this
reason, natural materials have been studied as alternative and safe
substitutes which can be used by both men and women.
In a preliminary study, an ethanol extract of Malva verticillata L.
(Malvaceae) seeds was selected as an activator of the Wnt pathway
by a Wnt/b-catenin reporter activity assay from a library of oriental herb extracts.
The seed of M. verticillata is an oriental medicine used as a diuretic, laxative and galactopoietic [15, 16], yet the bioactivities and
mechanisms of compounds isolated from M. verticillata seeds remain
poorly understood. In this study, we determined whether the seeds
of M. verticillata are effective modulators of the Wnt/b-catenin signal pathway in cultured human DPCs. Then, several extracts which
were further fractionated using various organic solvents were analysed for their effects on Wnt/b-catenin activity. Finally, the mechanism of action of myristoleic acid, which was identified as the active
compound of M. verticillata seeds, was studied.
Materials and methods
Cell culture
DPCs of human origin were purchased from PromoCell GmbH (Heidelberg, Germany) and were maintained in the appropriate Pro-
Silica gel C.C.
CH2Cl3/MeOH
61E
59B-7
Recrystal.
In cold MeOH
59E
59B-8
62A
59B-9
63A
Silica gel C.C.
CHCl3/MeOH/H2O
59B-10
Silica gel C.C.
CHCl3/MeOH/H2O
Silica gel C.C.
CHCl3/MeOH/H2O
62B
63B
63C
moCell growth medium. Immortalized DPCs (SDPC, SV40transformed DPCs) [17] were grown with Dulbecco’s modified
Eagle’s medium (DMEM, Hyclone, Logan, UT, U.S.A.) containing
10% (v/v) FBS (PAA Laboratories, Buckinghamshire, UK), 1% (v/v)
antibiotic–antimycotic (Gibco, NY, U.S.A.). Cells were incubated at
37°C in an atmosphere containing 5% CO2.
Extraction, isolation and structure determination of active
compounds
The seeds of M. verticillata were purchased at a market of oriental
herbs in Samsundang, Daejeon, Korea. The seeds of M. verticillata
(2.3 kg) were ground and extracted with ethanol for 6 h under
sonication (3 9 4 L). The macerate was concentrated in vacuo
(53.0 g) and partitioned to yield a methylene chloride extract
(27.0 g), ethyl acetate extract (2.7 g) and an aqueous extract
(23.0 g). The methylene chloride extract (27.0 g) was separated
by silica gel column chromatography eluting with a gradient system of n-hexane–EtOAc–MeOH–H2O (9.5: 0.5: 0: 0, 9: 1: 0: 0, 4:
1: 0:0, 1: 1: 0: 0, 0: 1: 0: 0, 0: 9: 1: 0, 0: 4: 1: 0, 0: 1:1: 0, 0:
0: 1: 0, 0: 0: 9.5: 0.5, v/v) to give ten fractions (59B-1~10).
59B-4 (2.6 g) was further purified using silica gel columns with
n-hexane–EtOAc (9.5 : 0.5, v/v) as an eluent to yield 60B
(200.0 mg).
We purified 59B-7 (0.9 g) by recrystallization from cold MeOH
to afford 59E (350.0 mg). We combined 59B-5 (0.8 g) and 59B-6
(0.6 g) for chromatography on a silica gel column eluting a mixture of CH2Cl2–MeOH (9.6: 0.4, v/v) to yield four fractions (F5AF5D). F5B (494.5 mg) was purified using silica gel column chromatography (n-hexane–EtOAc, 1: 1, v/v) to obtain 61E (40.0 mg).
We chromatographed 59B-8 (1.6 g) on a silica gel column eluting
a mixture of CHCl3–MeOH–H2O (7: 3: 0.3, v/v) to afford 62B
(340.0 mg). We subjected 59B-9 (1.6 g) to silica gel column chromatography with CHCl3–MeOH–H2O (7 : 3 : 0.3, v/v) to give 63C
(130.0 mg) and 63B (220.0 mg) (Fig. 1). The compounds of fractions were identified utilizing physical and spectral methods [16] as
well as by direct comparison of their data with the literature values
of b-sitosterol (60B) [18], verticilloside (61E) [16], daucosterol
(59E) [19], sucrose (62B) [20], raffinose (63C) [21] and myristoleic
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
International Journal of Cosmetic Science, 38, 148–154
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M. verticillata seed extracts upregulate the Wnt pathway
E. Y. Lee et al.
acid (63B) [22]. For the treatments, various fractions were dissolved in DMSO at a concentration of 10 mg mL 1.
Statistical analysis
Data are expressed as the mean SD. The statistical significance
was determined using Student’s t-test. P values < 0.01 were considered to be statistically significant.
Wnt/b-catenin reporter activity assay
Transfections were performed using Lipofectin reagent (Gibco)
according to the manufacturer’s instructions. Briefly, SDPCs
were grown to 50% confluency in 60-mm culture dishes using
growth medium. The Wnt/b-catenin reporter plasmid contained
a consensus TCF-binding element followed by the luciferase
gene. Plasmid DNA was diluted with Opti-MEM (Gibco), combined with Lipofectin reagent and then added to the culture
medium. Cells were replaced with fresh growth medium 6 h
after transfection and treated with various fractions of M. verticillata seeds or myristoleic acid (Sigma-Aldrich, St. Louis, MO,
U.S.A.) one day before the assay. Cell extracts were prepared
48 h after transfection. Luciferase activities were determined
using a luciferase assay system (Promega, Madison, WI, U.S.A.)
as previously described [17]. Experiments were performed in
triplicate.
Western blot analysis
DPCs were seeded in a 100-mm culture dish, pre-incubated for
24 h and then treated with test materials depending on the study
for 24 h. Treated DPCs were dissolved in PRO-PREP protein extraction solution (iNtRON Biotech, Korea). The proteins were separated
on 10% or 12% SDS-PAGE gels and transferred to 0.2 lm pore size
PVDF membranes (Millipore, Bedford, MA, U.S.A.). Membranes
were blocked and incubated with appropriate antibodies. Blots were
reacted with Immobilon Western reagent (Millipore) and detected
using an Amersham Hyperfilm electrochemiluminescence (ECL)
assay (GE Healthcare, Buckinghamshire, UK). Experiments were
performed twice.
Proliferation of DPCs
DPCs were plated at a density of 6000 cells/well in 48-well
plates with supplement-free medium. After attachment, cells were
incubated for 2 or 3 days in medium containing various concentrations of test materials. Cell proliferation was measured using
the EZ-CyTox assay kit (Daeil Lab Service, Korea). The absorbance was measured at 450 nm using a microplate reader (BioRad, Hercules, CA, U.S.A.). Cells were calculated by comparing
the optical density (OD) of each well to the values of authentic
standard curves. All the experiments were performed in triplicate.
Results
Malva verticillata seed extracts stimulate the Wnt/b-catenin
signalling pathway in cultured human DPCs
The effects of the ethanol extract of M. verticillata seeds on the
Wnt/b-catenin signalling pathway in cultured human DPCs were
tested. Because the reporter plasmid contained the firefly luciferase
gene under the control of a TCF-binding site, luciferase activity correlated with a stimulation of the Wnt/b-catenin signalling pathway. When DPCs were treated with an ethanol extract of
M. verticillata seeds, luciferase activities were increased up to twofold compared with the negative control group at 50 lg mL 1
(Fig. 2A). Because activation of the Wnt pathway stabilizes b-catenin through inhibition of its ubiquitination, we tested whether this
extract influenced intracellular b-catenin levels. Western blot analysis showed that the intracellular total b-catenin level was
increased by treatment with the M. verticillata seed extract
(Fig. 2B). The ethanol extract (59A) was fractionated into a methylene chloride extract (59B), an ethyl acetate extract (59C) and an
aqueous extract (59D). The activities of each fraction were tested
using a luciferase reporter assay. 59B significantly increased luciferase activity, whereas 59C and 59D showed no effect (data not
shown). 59B was further fractionated by silica gel column chromatography, and several compounds were isolated as described in
the materials and methods section. The fractions and compounds
were subjected to a luciferase activity assay. A noteworthy increase
in luciferase activity was detected when 63B was administered for
24 h (Fig. 3A). We also evaluated b-catenin accumulation by Western blot analysis after treatment of 63B with varying concentrations, confirming that 63B treatment significantly promoted the
activation of the Wnt/b-catenin pathway in a dose-dependent
RNA extraction and reverse transcription polymerase chain
reaction (RT-PCR)
Total RNA was extracted from DPCs that had been exposed to
myristoleic acid for 6 h using the Easy-spin total extraction kit
(iNtRON Biotech). cDNA synthesized from 2 lg total RNA was used
as the template for PCR. PCR amplification was performed with a
MyCyclerTM thermal cycler (Bio-Rad) as follows: a denaturing step
at 95°C for 5 min, 35–40 cycles at 95°C for 30 s each, annealing
temperature of 72°C for 30 s and final extension at 72°C for
5 min. The PCR products were run on a 2% agarose gel and visualized with a Gel DocTM EX Imager (Bio-Rad). Experiments were performed twice.
150
Figure 2 Malva verticillata seed extracts activate the Wnt/b-catenin pathway. (A) Activation of the Wnt/b-catenin pathway as measured by luciferase
reporter activity after treatment with vehicle or different concentrations of
unfractionated (ethanol) extract of M. verticillata seeds for one day.
**P < 0.01 compared to control. (B) Expression of b-catenin by Western blot
showing that the ethanol extract of M. verticillata seeds (50 lg mL 1)
increased the amount of intracellular b-catenin in cultured DPCs compared
to vehicle controls. Numbers on the bottom of b-catenin bands represent
densitometric quantitation relative to controls.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
International Journal of Cosmetic Science, 38, 148–154
M. verticillata seed extracts upregulate the Wnt pathway
E. Y. Lee et al.
A
B
0 μg mL–1
10 μg mL–1
20 μg mL–1
Figure 3 The 63B compound of Malva verticillata seeds significantly activated the Wnt/b-catenin pathway. (A) Activation of the Wnt/b-catenin
pathway as measured by luciferase reporter activity after treatment with
various fractions of M. verticillata seeds for 24 h. **P < 0.01 compared to
vehicle control. (B) Expression of b-catenin by Western blot showing that
63B accumulates at the intracellular protein level in cultured DPCs at all
tested concentrations. Numbers on the bottom of b-catenin bands represent
densitometric quantitation relative to vehicle controls.
manner (Fig. 3B). From the above results, we decided to focus on
the action of the 63B fraction of M. verticillata seeds.
Myristoleic acid, an active compound in Malva verticillata seeds,
stimulates the Wnt/b-catenin signalling pathway
The structure of the active compound was identified as myristoleic
acid by 1H-NMR and 13C-NMR [16]. Therefore, we next tested
whether commercially available myristoleic acid showed a similar
effect as 63B on human cultured DPCs through the Wnt/b-catenin
reporter system. The Wnt reporter activity was increased 1.10-,
1.95- and 3.04-fold by treatment with myristoleic acid at 10, 50
and 100 lg mL 1, respectively (P < 0.01) (Fig. 4A). This result
was confirmed with the above result by 63B treatment; the stimulated level was not significantly different following treatment with
63B or myristoleic acid.
Dermal papilla size is well-correlated with the hair cycle phase,
and the cell population of dermal papilla increases in the anagen
phase [23]. For this reason, the effect on DPC proliferation is frequently investigated during the development of new therapies to
treat hair loss. Myristoleic acid stimulated the proliferation of
cultured DPCs in a dose-dependent manner up to 20 lg mL 1
(P < 0.01), but it was cytotoxic at higher concentrations
(Fig. 4B).
Effect of myristoleic acid on the expression of cytokines related to
hair growth
Various growth factors such as IGF-1, KGF, VEGF and HGF are
involved in the regulation of hair morphogenesis and hair growth.
50 μg mL–1
Figure 4 Myristoleic acid stimulates luciferase activity and the proliferation
of cultured human DPCs. (A) Myristoleic acid, an active compound of 63B,
stimulates Wnt reporter activity in a dose-dependent manner. **P < 0.01
compared to vehicle control. (B) Cell proliferation of DPCs measured by the
WST-based assay after treatment with different concentrations of myristoleic
acid for 2 or 3 days. Myristoleic acid stimulates proliferation at concentrations up to 20 lg mL 1. **P < 0.01 compared to vehicle control.
The expression levels of IGF-1, KGF, VEGF and HGF in cultured
DPCs were significantly increased by myristoleic acid treatment.
Although the mRNA levels of VEGF and HGF were increased in a
dose-dependent fashion, those of IGF-1 and KGF were increased
only at 50 lg mL 1 (Fig. 5A). The mRNA levels of IGF-1 and KGF
could also possibly be increased at lower doses, but this was not
detected.
Effect of myristoleic acid on signalling molecules
The effects of myristoleic acid on various signalling pathways associated with the regulation of the hair cycle and hair growth were
monitored by Western blot analysis. Western blots showed that the
phosphorylation of p-38, CREB and Akt was significantly
enhanced, whereas ERK was slightly increased after treatment with
myristoleic acid (Fig. 5B). The upregulation of phosphorylated
p-38, CREB and Akt strongly indicated that myristoleic acid is a
novel modulator of the Wnt pathway, although the mechanism of
signal transduction by myristoleic acid requires further investigation.
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
International Journal of Cosmetic Science, 38, 148–154
151
M. verticillata seed extracts upregulate the Wnt pathway
A
E. Y. Lee et al.
B
Figure 5 The effect of myristoleic acid on the expression of cytokines and
signalling molecules. (A) Semi-quantitative RT-PCR for the mRNA levels of
the growth factors VEGF, KGF, IGF-1 and HGF after treatment with vehicle
or myristoleic acid. (B) Western blot for p-38, CREB and Akt phosphorylation in cultured human DPCs after treatment with vehicle or myristoleic acid
at different concentrations for 6 h.
Discussion
The interest in treating hair loss has increased in recently in parallel with an increasing number of hair loss patients. Therefore, it is
very important to develop new therapeutic agents to prevent hair
loss. M. verticillata seeds have been used as diuretic, laxative and
galactopoietic medicines [15]; however, the active compounds and
their modes of action remain largely unknown. Moreover, the effect
of M. verticillata seeds on hair follicles has not been reported until
now. In this study, we demonstrated that an extract of M. verticillata seeds activated the Wnt/b-catenin signal pathway in human
cultured DPCs and immortalized human DPCs. Myristoleic acid was
identified as an active compound and stimulated the proliferation
of DPCs and induced transcription of IGF-1, KGF, VEGF and HGF.
It also enhanced phosphorylation of Akt, CREB and p38. The
obtained results indicate that myristoleic acid, as a stimulating
agent of M. verticillata seeds, might be an effective treatment for
hair loss through the activation of the Wnt/b-catenin signalling
pathway and subsequent cell proliferation and transcription of hair
growth-related cytokines in cultured human DPCs. The biological
activities of myristoleic acid have been studied by a number of
investigators [24, 25]. Myristoleic acid stimulates leucocyte maturation and induces apoptosis and necrosis in prostatic carcinoma
[26]. Interestingly, myristoleic acid was reported to inhibit flank
organ growth in a hamster model by inhibiting 5-alpha-reductase
[27], yet the effect of myristoleic acid on hair growth has not been
investigated until now. This is the first report on the role of myristoleic acid on hair growth.
The Wnt/b-catenin pathway plays a pivotal role in hair morphogenesis and regeneration. The Wnt family includes 19 members that
comprise two pathways. The canonical Wnt/b-catenin signalling
pathway contributes to promotion of hair follicle growth, morphogenesis and regeneration. The non-canonical Wnt signalling pathway is described as the canonical pathway antagonist, and it acts
152
through a different route [28]. In the canonical signalling pathway,
the Wnt ligand binds to the Frizzled receptor and then activates
dishevelled, which induces phosphorylation of GSK3b. As a result,
b-catenin phosphorylation by GSK3b is inhibited and b-catenin is
stabilized. Accumulation of b-catenin in the cytosol leads to translocation into the nucleus where it interacts with the TCF/LEF transcription factors to activate its target genes [29]. Aberrant activation
of the Wnt pathway promotes uncontrolled cell growth and survival, and can consequently drive cancer formation. Drugs developed to block aberrant Wnt signalling have great potential as
effective cancer therapeutics, but their prolonged use might carry
have side effects in organs such as the skin, where Wnt pathway
activity is pivotal for tissue regeneration [30]. Our experimental data
showed that myristoleic acid, as an active compound isolated from a
methylene chloride fraction of M. verticillata seeds, upregulated Wnt
reporter activity and stabilized b-catenin. Therefore, this oriental
herb extract may be a good candidate for the treatment of hair loss,
although current therapies targeting the Wnt pathway must first be
considered. Previous studies reported that IGF-1, KGF, VEGF and
HGF are involved in hair growth; this study found that myristoleic
acid treatment significantly increased the expression of these growth
factors in cultured DPCs. IGF-I stimulated hair growth in a dose-dependent manner and prevented hair follicles from entering the catagen phase [31, 32]. Recombinant KGF induces hair growth at the
injection sites [33], and VEGF has been shown to control hair
growth and follicle size by angiogenesis in a transgenic mouse model
[34]. HGF stimulates follicle growth and DNA synthesis in both
human hair and mouse vibrissae [35]. Because it stimulates secretion of these growth factors, myristoleic acid might be an effective
treatment for hair loss by prolonging the anagen phase and promotion of hair regrowth.
Phosphorylation of ERK and Akt increases the b-catenin/TCF
complex through phosphorylation of GSK3b. In other words, the
Wnt/b-catenin signalling pathway is in an activate state when the
ERK and/or Akt signalling pathways are activated [36, 37]. p38
MAPK can regulate the Wnt signalling pathway through inactivation of GSK3b via canonical Wnt/b-catenin signalling [38, 39].
p38 MAPK phosphorylation can also regulate the non-canonical
Wnt signalling pathway independently through dishevelled [40].
CREB is a direct downstream effector of Wnt signalling via adenylyl
cyclase and protein kinase A. The Wnt protein also induces phosphorylation of CREB [41]. In this study, the upregulation of phosphorylated p38, CREB and Akt strongly indicated that myristoleic
acid is a novel modulator of the Wnt pathway. The mechanism of
action of signal transduction by myristoleic acid needs to be investigated further.
In summary, myristoleic acid, an active compound in M. verticillata seeds, activated Wnt reporter activity and increased cell proliferation in cultured human DPCs. This is the first study to report
the bioactivity of a compound isolated from M. verticillata seeds for
the treatment of hair loss. This oriental herb extract may be a good
candidate for treatment of hair loss by modulating the Wnt/b-catenin pathway in DPCs.
Acknowledgement
This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of
Korea (Grant No. HN12C0053).
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
International Journal of Cosmetic Science, 38, 148–154
M. verticillata seed extracts upregulate the Wnt pathway
E. Y. Lee et al.
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