Download LPAproducing enzyme PAPLA1 regulates hair follicle development

The EMBO Journal (2011) 30, 4248–4260
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2011 European Molecular Biology Organization | All Rights Reserved 0261-4189/11
THE
EMBO
JOURNAL
LPA-producing enzyme PA-PLA1a regulates hair
follicle development by modulating EGFR
signalling
Asuka Inoue1,2,*, Naoaki Arima1,
Jun Ishiguro1, Glenn D Prestwich3,
Hiroyuki Arai2,4 and Junken Aoki1,5,*
1
Laboratory of Molecular and Cellular Biochemistry, Graduate School
of Pharmaceutical Sciences, Tohoku University, Sendai, Japan,
2
Department of Health Chemistry, Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Tokyo, Japan, 3Department of
Medicinal Chemistry, The University of Utah, Salt Lake City, UT, USA,
4
CREST, Japan Science and Technology Corporation, Tokyo, Japan and
5
PRESTO, Japan Science and Technology Corporation, Tokyo, Japan
Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPAproducing enzyme, phosphatidic acid-selective phospholipase A1a (PA-PLA1a, also known as LIPH), and a recently
identified LPA receptor, P2Y5 (also known as LPA6).
However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle
development. PA-PLA1a-deficient mice generated in this
study exhibited wavy hairs due to the aberrant formation
of the inner root sheath (IRS) in hair follicles, which
resembled mutant mice defective in tumour necrosis factor
a converting enzyme (TACE), transforming growth factor a
(TGFa) and EGFR. PA-PLA1a was co-localized with TACE,
TGFa and tyrosine-phosphorylated EGFR in the IRS. In PAPLA1a-deficient hair follicles, cleaved TGFa and tyrosinephosphorylated EGFR, as well as LPA, were significantly
reduced. LPA, P2Y5 agonists and recombinant PA-PLA1a
enzyme induced P2Y5- and TACE-mediated ectodomain
shedding of TGFa through G12/13 pathway and consequent
EGFR transactivation in vitro. These data demonstrate that a
PA-PLA1a–LPA–P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE–TGFa–EGFR pathway,
thus underscoring the physiological importance of LPAinduced EGFR transactivation.
The EMBO Journal (2011) 30, 4248–4260. doi:10.1038/
emboj.2011.296; Published online 19 August 2011
Subject Categories: signal transduction; development
Keywords: EGFR transactivation; hair follicle;
lysophosphatidic acid (LPA); transforming growth factor a
(TGFa); tumour necrosis factor a converting enzyme (TACE)
*Corresponding authors. A Inoue or J Aoki, Laboratory of Molecular and
Cellular Biochemistry, Graduate School of Pharmaceutical Sciences,
Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
Tel.: þ 81 22 795 6861; Fax: þ 81 22 795 6859;
E-mail: [email protected] or
Tel.: þ 81 22 795 6860; Fax: þ 81 22 795 6859;
E-mail: [email protected]
Received: 20 May 2011; accepted: 20 July 2011; published online:
19 August 2011
4248 The EMBO Journal VOL 30 | NO 20 | 2011
Introduction
Lysophosphatidic acid (1- or 2-acyl-lysophosphatidic acid;
LPA) is a bioactive lipid with numerous biological functions.
LPA exerts most of its functions through G protein-coupled
receptors (GPCRs) (Anliker and Chun, 2004; Moolenaar et al,
2004). So far, six LPA-specific GPCRs belonging to either the
endothelial cell differentiation gene family (LPA1,2,3) or P2Y
family (LPA4,5,6) have been identified (Anliker and Chun,
2004; Ishii et al, 2009; Chun et al, 2010). These LPA receptors,
by coupling with different G proteins and having distinct
expression patterns, play important roles in various physiological and pathophysiological conditions (Aoki et al, 2008;
Skoura and Hla, 2009; Choi et al, 2010).
Extracellular LPA production occurs at least in two distinct
pathways (Aoki et al, 2008). In biological fluids such as
serum, a soluble enzyme called autotaxin (ATX) hydrolyzes
lysophospholipids with its lysophospholipase D activity and
produces LPA (Aoki et al, 2002). In cellular membranes,
membrane-associated enzymes called phosphatidic acid
(PA)-selective phospholipase A1a (PA-PLA1a, also known as
mPA-PLA1a and LIPH) (Sonoda et al, 2002) and PA-PLA1b
(also known as mPA-PLA1b and LIPI) (Hiramatsu et al, 2003)
hydrolyze PA and produce 2-acyl-LPA (with acyl chain at the
sn-2 position of glycerol). While the functions of ATX have
been well characterized in angiogenesis (Tanaka et al, 2006;
van Meeteren et al, 2006), brain development (Koike et al,
2009), cancer metastasis (Mills and Moolenaar, 2003) and
lymphocyte traffic (Kanda et al, 2008), the physiological roles
of the latter enzymes have remained unclear until recently.
Kazantseva et al (2006) reported that homozygous mutation in the PA-PLA1a gene was found to cause a congenital
hair disorder in human (termed LAH2). Affected individuals
were characterized by hereditary woolly hair and/or sparse
hair (Kazantseva et al, 2006; Shimomura et al, 2009).
Interestingly, homozygous mutations in the P2Y5 (also
known as P2RY5) gene were found to be responsible for
another congenital hair disorder (termed LAH3) (Pasternack
et al, 2008; Shimomura et al, 2008). The P2Y5 gene encodes
an orphan GPCR sharing 56% amino-acid identity with LPA4,
which is presently the closest known homologue of P2Y5.
Accordingly, Yanagida et al (2009) showed that LPA behaved
as a ligand for P2Y5 and thus named P2Y5 as LPA6. Thus, we
postulated that PA-PLA1a hydrolyzes PA on the plasma membrane and provides 2-acyl-LPA for P2Y5, which leads to hair
follicle formation (Figure 1A). The idea that PA-PLA1a and
P2Y5 have cooperative roles in hair follicle formation is also
supported by observations that (i) both PA-PLA1a and P2Y5
are highly expressed in hair follicles, especially in epithelial
cells (Kazantseva et al, 2006; Shimomura et al, 2008) and (ii)
affected individuals with LAH2 and LAH3 are clinically indistinguishable. It is likely that PA-PLA1a activates P2Y5 through
production of LPA. However, how LPA-P2Y5 signalling regulates hair follicle formation remains to be determined. In this
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LPA-induced EGFR transactivation in hair follicles
A Inoue et al
B
A
C
+/+
+/+
–/–
–/–
Phosphatidic acid (PA)
PA-PLA1α
Lysophosphatidic acid (LPA)
P2Y5
Epithelial cell
D
E
+/+
–/–
+/+
Hair formation
F
+/+
–/–
–/–
Figure 1 Wavy hair phenotype of PA-PLA1a/ mice. (A) Scheme of PA-PLA1a and P2Y5 pathway in hair formation, proposed by human
genetic studies. Genetically, hereditary mutations in the PA-PLA1a gene (Kazantseva et al, 2006; Shimomura et al, 2009) and the P2Y5 gene
(Pasternack et al, 2008; Shimomura et al, 2008) cause clinically indistinguishable hair disorders in human. Biochemically, PA-PLA1a is capable
of hydrolyzing PA (Sonoda et al, 2002) and P2Y5 responds to LPA (Yanagida et al, 2009). It remained unclear whether LPA was actually
involved in this pathway and, if so, how LPA signalling regulated hair formation. (B, C) Hair coats (B) and vibrissa hairs (C) of WT ( þ / þ ) and
PA-PLA1a/ (/) mice on postnatal day (P) 25. Note that hair coat of a WT mouse shows shiny appearance, whereas hair coat of a PAPLA1a/ mouse shows wavy and matted appearance. (D–F) Pelage hairs were observed under an optical microscope (D, E) and a scanning
electron microscope (E). Compared with WT hairs, all of four hair types of PA-PLA1a / pelage hairs (inset from top: guard, awl, auchene and
zigzag) show waviness (D). Note that PA-PLA1a/ hair shows disorganized medulla and irregular melanin piles (E) and wavy cuticle (F).
Scale bars, 2 cm (B), 500 mm (D), 20 mm (E) and 5 mm (F).
study, to obtain clues about the molecular mechanism underlying LPA-mediated hair follicle formation, we generated and
analysed PA-PLA1a-deficient (PA-PLA1a/) mice and found
that epidermal growth factor receptor (EGFR) signalling underlies LPA-mediated hair follicle formation.
Results
PA-PLA1a/ mice show wavy hair phenotype
To investigate the effect of PA-PLA1a deletion in mice, we
disrupted the PA-PLA1a gene of murine embryonic stem cells
by replacing exon 3, which encodes the active residue Ser154,
with a neomycin resistance cassette (Supplementary Figure
S1A). Deletion of exon 3 resulted in a frameshift mutation
and no immunoreactive PA-PLA1a protein (Supplementary
Figure S1C; Figures 3F and 4D). PA-PLA1a/ mice were
born with the expected Mendelian ratio from an intercross
of heterozygous parents ( þ / þ : þ /:/ ¼ 86:205:84).
PA-PLA1a/ mice were distinguishable from wild-type
(WT) or heterozygous littermates by aberrant hair characteristics including wavy vibrissa hair and a matted coat
(Figure 1B and C). Pelage hairs from PA-PLA1a/ mice
showed waviness, disorganized medulla, irregular melanin
piles and wavy cuticles (Figure 1D–F). The hair abnormalities
were observed in all PA-PLA1a/ mice (4300 animals
observed) and, more importantly, in two PA-PLA1a/ lines
derived from independent ES clones (clones 1D-A1 and
4C-D3). Thus, in the following experiments, only the line
derived from clone 1D-A1 was examined. The wavy hair
phenotype was most apparent from 1 to 4 weeks of age and
& 2011 European Molecular Biology Organization
became less severe after the start of the anagen (growth)
phase of the first hair cycle. Of note, these hair abnormalities
of PA-PLA1a/ mice resembled those of mutant mice defective in keratin genes specific to the inner root sheath (IRS),
the layer surrounding the hair shaft (Figure 3E), of hair
follicles (Kikkawa et al, 2003; Peters et al, 2003; Tanaka
et al, 2007), as well as transforming growth factor a
(TGFa)-related genes including TNFa-converting enzyme
(TACE, also known as ADAM17) (Peschon et al, 1998),
TGFa (Luetteke et al, 1993; Mann et al, 1993) and EGFR
(Luetteke et al, 1994), as discussed below. Unlike humans
with PA-PLA1a-null mutations, PA-PLA1a/ mice did not
seem to develop alopecia. Besides hair abnormalities, we
could not find any difference between PA-PLA1a/ mice
and WT or heterozygous littermates such as fertility or
growth rate under normal conditions (data not shown).
Reduced 2-acyl-LPA in PA-PLA1a/ mice
LPA detected in vivo consists of a mixture of LPA species
with various fatty acids attached to either the sn-1 (1-acylLPA) or sn-2 (2-acyl-LPA) position of the glycerol backbone.
To examine which LPA species, if any, are produced by
PA-PLA1a in skin and hair follicles, which generate hair
shafts, we analysed LPA species of tissue extracts using a
newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. LPA ions were selectively
detected by the selective reaction monitoring mode (e.g.
m/z 409.3 for the negatively charged parent ion of 16:0-LPA
and m/z 153.0 for the fragment ion of 16:0-LPA). Consistent
with a previous reversed-phase LC condition (Creer and
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A Inoue et al
phosphatidylglycerol), LPI (lysophosphatidylinositol), LPS
(lysophosphatidylserine) and S1P (sphingosine 1-phosphate)
were unchanged in both isolated vibrissa hair follicles and
dorsal skin of PA-PLA1a/ mice (Figure 2C; Supplementary
Figure S2B and C and data not shown), confirming that LPA
was specifically decreased in PA-PLA1a/ mice. These data
demonstrate that PA-PLA1a produces LPA, especially 2-acylLPA with unsaturated fatty acids, in hair follicle.
Gross, 1985), we observed two separate peaks corresponding
to the 2-acyl isomer (first elution) and the 1-acyl isomer
(second elution) (Supplementary Figure S2A). Among eight
LPA species examined, five of six unsaturated LPA species
(18:2, 18:1, 20:5, 20:4 and 22:6) were significantly reduced in
both isolated vibrissa hair follicles and dorsal skin of PAPLA1a/ mice (Figure 2A and data not shown). Notably, in
hair follicle of PA-PLA1a/ mice, the six unsaturated LPA
species were reduced by 490%, whereas saturated LPA
species were unchanged (18:0) or slightly decreased (16:0)
(Figure 2A). When 1-acyl isomer and 2-acyl isomer of LPA
were separately quantified, 2-acyl-LPA in three predominant
LPA species (16:0, 18:2 and 18:1) were significantly lowered
in PA-PLA1a/ hair follicles, whereas 1-acyl-LPA remained
largely unchanged (Figure 2B). These data are consistent
with the asymmetry of fatty acid composition of phospholipids in which saturated fatty acid and (poly)unsaturated fatty
acid are generally attached at the sn-1 and sn-2 positions of
the glycerol backbone, respectively. The levels of six
other lysophospholipids including LPC (lysophosphatidylcholine), LPE (lysophosphatidylethanolamine), LPG (lyso-
Impaired IRS formation in PA-PLA1a/ hair follicles
Among hair follicle layer-specific keratins, wavy hair phenotype appears only in mice with mutations for IRS-specific
keratins (Krt25, Krt27 and Krt71) (Kikkawa et al, 2003; Peters
et al, 2003; Tanaka et al, 2007). Thus, the irregular hair in
PA-PLA1a/ mice occurred most likely due to defects in
the formation of the IRS. Indeed, histological analysis of cross
sections of vibrissa hair follicles with haematoxylin and eosin
(HE) showed that the IRS of PA-PLA1a/ hair follicles,
compared with that of WT hair follicles, was irregularly
structured (Figure 3A). Other hair follicle structures such as
the outer root sheath (ORS) and the dermal papilla appeared
Relative peak area
A 0.07
+/+
0.05
0.04
0.03
0.02
0.01
**
0
16:0
***
18:2
B
Relative peak area
–/–
0.06
0.06
0.05
0.04
0.03
0.02
0.01
0
***
NS
18:1
18:0
+/+
–/–
**
20:4
**
20:5
0.01
Relative peak area
*
22:5
NS
0.008
0.006
NS
0.004
**
16:0
***
18:2
***
18:1
NS
NS
0.002
0
18:0
16:0
2-acyl-LPA
C
**
22:6
18:1
18:0
1-acyl-LPA
+/+
2
**
18:2
–/–
1.5
1
0.5
0
**
LPA
LPC
LPE
LPG
LPI
LPS
S1P
Figure 2 LPA is lowered in PA-PLA1a/ hair follicles. (A) Relative abundance of eight LPA species in vibrissa hair follicles. Lipids in vibrissa
hair follicles from P15 WT ( þ / þ ) and PA-PLA1a/ (/) mice (during the hair growth phase) were extracted with methanol and analysed
by an LC-MS/MS method. Peak areas of 1-acyl-LPA and 2-acyl-LPA were combined and normalized to the internal standard 17:0-LPA (n ¼ 8).
Numbers in the x axis denote acyl chains that are attached to LPA. 16:0, palmitoyl; 18:2, linoleoyl; 18:1, oleoyl; 18:0, stearoyl; 20:5,
eicosapentanoyl; 20:4, arachidonoyl; 22:6, docosahexanoyl; 22:5, docosapentaenoyl. (B) Same as (A), but 2-acyl-LPA and 1-acyl-LPA in (A)
were separately quantified. (C) LC-MS/MS analysis of lysophospholipids in vibrissa hair follicles. Except for S1P, eight acyl moieties of each
lysophospholipid were monitored and the sum of the peak areas was used for quantification (see Supplementary Figure S2 for details).
The mean value in þ / þ sample was set at 1 (n ¼ 8). Asterisks, *Po0.05, **Po0.01, ***Po0.001 versus þ / þ . NS, not significant difference
between þ / þ and /.
4250 The EMBO Journal VOL 30 | NO 20 | 2011
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LPA-induced EGFR transactivation in hair follicles
A Inoue et al
Henle’s layer
+/+
I
–/–
α
1
LA
α
A1
PL
I
P
PA
P
DA
PA
P
DA
5
Cortex
Medula
F
Hair cuticle
IRS cuticle
Huxley’s layer
Hair shaft
K8
K7
5
Tr
ic
ho
hy
al
in
K7
2
K1
7
ORS
–/–
IRS
Henle’s layer
+/+
A
Companion layer
E
K71
Huxley’s layer
IRS cuticle
Relative mRNA
expression
B
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
+/+
G
*
Gata3
K
**
Tchh
Krt71
Krt72
IRS
K85
+/+
–/–
72
**
**
Krt85
Hair shaft
C
–/–
K71
+/+
–/–
Krt75
Companion
layer
K72
+/+
–/–
Krt17
ORS
LA
P
P
A-
α
in
al
1
hy
Tr
o
ich
e
M
g
er
H
75
K
LA
P
A-
P
α
in
al
1
hy
Tr
o
ich
ge
er
M
Vim
Dermal
sheath
K75
+/+
–/–
Keratin
α-tubulin
+/+
–/–
*
**
K85
Hair shaft
K71
K72
IRS
K75
HE
Relative protein
expression
1.4
1.2
1
0.8
0.6
0.4
0.2
0
In situ
hybridization
I
D
Companion layer
Figure 3 Impaired IRS formation in PA-PLA1a/ hair follicles. (A) HE staining of vibrissa hair follicles. Magnified views of the boxed areas are
shown in the bottom panels. The arrow indicates the discontinuous Henle’s layer in a PA-PLA1a/ vibrissa hair follicle. Scale bars, 20 mm (top)
and 5 mm (bottom) (B) Quantitative RT–PCR (qRT–PCR) analysis of hair follicle layer-specific markers in P4 dorsal hair follicles. The number of
transcripts was normalized to the housekeeping gene, Gapdh, in the same sample and the mean value in þ / þ sample was set at 1 (n ¼ 4).
(C, D) Immunoblot analysis of hair follicle layer-specific markers in P10 dorsal skin and densitometric quantification. The marker intensity was
normalized to a-tubulin in the same sample and the mean value in þ / þ sample was set at 1 (n ¼ 4). (E) A schematic illustration of hair
follicle layers and marker expressions in follicular layers. (F–H) Immunofluorescent detection of PA-PLA1a in dorsal hair follicles. Nuclei were
stained with DAPI. Specificity of anti-PA-PLA1a antibody was confirmed using PA-PLA1a/ hair follicle (F). K72 (G) and K75 (H) (detected
with Alexa Fluor 647) were pseudo-coloured ‘blue.’ Magnified views of the boxed areas are shown in the bottom panels. The arrows indicate
co-expression of PA-PLA1a with K72 (G) and K75 (H) in WT hair follicles. Scale bars, 20 mm (top) and 5 mm (bottom). (I) In situ hybridization of
PA-PLA1a mRNA in P15 WT vibrissa hair follicles (upper panels, purple hybridization signal). A serial section was stained with HE (lower
panels). No significant signals were detected using a sense probe (data not shown). Note that brown dots inside the hair cuticle in the middle
panel are melanin granules, not a hybridization signal. Left panels, longitudinal sections of hair follicles; middle panels, enlarged images of the
boxed areas in the left panels; right panels, cross sections of hair follicles. Arrows, IRS; arrowheads, hair cuticle. Scale bars represent 100 mm in
left panels and 20 mm in middle and right panels. *Po0.05, **Po0.01 versus þ / þ .
normal (Figure 3A and data not shown). The IRS consists of
the three layers (Henle’s layer, Huxley’s layer and the IRS
cuticle, from outermost to innermost; Figure 3E) and plays an
important role in anchoring hair shaft and supporting of its
development. In WT hair follicles, Henle’s layer was recognized by fully keratinized and continuous cells that were
unstained by HE, and Huxley’s layer consisted of multiplelayered cells containing Eosin-positive trichohyalin granules
(Figure 3A). In contrast, in PA-PLA1a/ hair follicles,
Henle’s layer appeared to be discontinuous and Huxley’s
layer protruded into the ORS (Figure 3A). We noticed, however, that the IRS length was not significantly different
between the two genotypes (Supplementary Figure S3A–D),
& 2011 European Molecular Biology Organization
suggesting that the IRS in PA-PLA1a/ hair follicle, although
morphologically deformed, could still undergo maturation/
keratinization processes. The hair cycle and epidermal structure were also unaffected in PA-PLA1a/ mice (data not
shown). To confirm IRS malformation at the biochemical
level, we next examined expressions of layer-specific marker
genes. Quantitative RT–PCR revealed remarkable reductions
in IRS markers (Krt71, Krt72 and Tchh encoding Keratin 71
(K71), K72, trichohyalin, respectively) (Langbein et al, 2003,
2006) as well as a slight reduction in a companion layer
marker (Krt75) in PA-PLA1a/ hair follicles (Figure 3B).
Expression levels of other hair follicle markers including
hair shaft (Krt85), ORS (Krt17) or dermal sheath (Vim
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encoding Vimentin) (Schweizer et al, 2007) were not different
between the two genotypes (Figure 3B). Notably, the expression of an early IRS differentiation marker (Gata3) (Kaufman
et al, 2003) was not affected in PA-PLA1a/ mice
(Figure 3B), in agreement with the existence of IRS structures
(Figure 3A). At the protein level, IRS markers (K71 and K72)
were also significantly decreased in PA-PLA1a/ hair follicles whereas a hair shaft marker (K85) and a companion
layer marker (K75) were not (Figure 3C and D). These data
again suggest that, among the multiple layers in hair follicles,
the IRS is specifically affected by disruption of PA-PLA1a,
which may lead to impaired guidance and maturation of
developing hair shafts, thus resulting in wavy hair formation.
In situ localization of PA-PLA1a expression in the hair
follicles was analysed by immunofluorescent staining and
in situ mRNA hybridization techniques. Immunofluorescent
staining detected the presence of PA-PLA1a protein in hair
follicle epithelial cells (Figure 3F). The PA-PLA1a signals were
not observed in PA-PLA1a/ hair follicles (Figure 3F), confirming the specificity of the anti-PA-PLA1a rat monoclonal
antibody established in this study. We also performed double
immunostaining using anti-PA-PLA1a and hair follicle layerspecific markers. An intense PA-PLA1a signal within the hair
follicles was co-localized in two layers with an IRS
cuticle marker (K72) and a companion layer marker (K75)
(Figure 3G and H). The localization of PA-PLA1a was also
confirmed by an in situ mRNA hybridization analysis, which
showed that an intense mRNA signal in the IRS and a less
intense signal in the companion layer (Figure 3I). This
expression pattern was consistent with the observation
that PA-PLA1a/ hair follicles exhibited aberrant IRS
(Figure 3A–D), strongly suggesting that the primary defect
in PA-PLA1a/ hair follicle is in the IRS.
PA-PLA1a is co-expressed with TACE, TGFa and
EGFR in hair follicles
We noticed that the wavy hair phenotype of PA-PLA1a/
mice was similar to the phenotypes of TACE-deficient
(Peschon et al, 1998), TACE mutant (known as woe)
(Hassemer et al, 2010), TGFa-deficient, TGFa mutant
(known as wa-1) (Luetteke et al, 1993; Mann et al, 1993)
and EGFR mutant (known as wa-2) mice (Luetteke et al,
1994). TACE, a member of a disintegrin and metalloprotease
(ADAM) family, is capable of cleaving a transmembrane
proform of TGFa (Sahin et al, 2004), a process referred to
as ectodomain shedding, and the subsequent binding of
soluble TGFa to EGFR results in activation of EGFR, known
as transactivation. Thus, the TACE–TGFa–EGFR pathway has
been recognized as an important regulator of hair follicle
development. The phenotypic resemblance of these mutant
mice to PA-PLA1a/ mice led us to hypothesize that the
TACE–TGFa–EGFR pathway is regulated by PA-PLA1a, most
likely through production of LPA and the subsequent activation of P2Y5 in hair follicles. Immunofluorescent staining
revealed that TACE, TGFa and the phosphorylated form of
EGFR (p-EGFR; phosphorylated at Tyr1068, a critical site for
autophosphorylation), were co-expressed with PA-PLA1a in
the IRS, specifically in the IRS cuticle of the K72-positive layer
(Figure 4A–C). We found that PA-PLA1a was upregulated
during the growth (morphogenesis or anagen) phase of the
hair cycle and downregulated during the regression (catagen)
and resting (telogen) phases at both the protein and mRNA
4252 The EMBO Journal VOL 30 | NO 20 | 2011
levels (Figure 4D–F). The expression pattern of P2Y5 was
also hair cycle dependent and was nearly identical to that of
PA-PLA1a in both the developmental and adult stages
(Figure 4F; Supplementary Figure S4), suggesting that both
P2Y5 and PA-PLA1a have cooperative roles during the anagen
phase. Our results are consistent with previous observations
that TGFa and P2Y5 were expressed in the IRS (Luetteke et al,
1993; Shimomura et al, 2008). These data strongly support
our hypothesis that PA-PLA1a and P2Y5 regulates hair follicle
formation through the TACE–TGFa–EGFR pathway.
Impaired TGFa-EGFR signalling in PA-PLA1a/
hair follicles
To test this hypothesis, we first examined the level of TGFa
in PA-PLA1a/ hair follicles. The soluble TGFa was
significantly lower (by 61% reduction) in PA-PLA1a/ hair
follicles than in WT hair follicles (Figure 4G), whereas TGFa
mRNA expression and membrane-bound TGFa protein
were not significantly different between the two genotypes
(Supplementary Figure S5A and B). Immunofluorescent
staining also showed no apparent differences in the TGFa
expression patterns (Supplementary Figure S6B). Immunoprecipitation and immunoblot analyses of dorsal skin lysate
revealed that tyrosine-phosphorylated EGFR (i.e. activated
EGFR) was lowered in PA-PLA1a/ mice than in WT mice
(Figure 4H and I). Immunofluorescent staining using an
antibody against p-EGFR confirmed that p-EGFR signal in
the IRS was reduced in PA-PLA1a/ hair follicles (Figure 4J).
These results indicate that PA-PLA1a induces TGFa release
and subsequent EGFR phosphorylation in vivo, which is
required for the proper development of hair follicles.
P2Y5 and TACE mediate LPA-induced TGFa release
and EGFR transactivation in keratinocytes
LPA and other GPCR agonists have been shown to induce
transactivation of EGFR in various cell types in vitro (Daub
et al, 1997). A critical step of EGFR transactivation is ADAMmediated ectodomain shedding of EGFR ligands such as
TGFa and heparin-binding EGF-like growth factor (HB-EGF)
(Peschon et al, 1998; Prenzel et al, 1999; Sahin et al, 2004).
We first examined whether LPA induces TGFa release and
EGFR transactivation in keratinocytes. When primary epidermal keratinocytes were treated with LPA (18:1), the cells
released significant amounts of TGFa (Figure 5A), which was
equivalent to the amount observed in cells treated with 12-Otetradecanolyphorbol-13-acetate (TPA) (Figure 5A), a wellestablished PKC-dependent ADAM activator. The LPA-induced TGFa release was completely blocked by pretreatment
with a broad-spectrum metalloprotease inhibitor, SDZ 242–
484 (Kottirsch et al, 2002) (Figure 5A). Among the LPA
species tested, LPA (20:4) was the most potent in inducing
TGFa release in keratinocytes (Figure 5B). Interestingly, we
found that LPA species that were abundantly present in
hair follicles (16:0, 18:1 and 20:4) (Figure 2A) had higher
ability to induce TGFa release (Figure 5B). In the primary
keratinocytes, LPA also induced an increase in the level of
phosphorylated EGFR at Tyr1068 (p-EGFR) as well as phosphorylated ERK1/2 (p-ERK1/2), one of the downstream
targets of EGFR (Figure 5C). Both p-EGFR and p-ERK were
attenuated by pretreatment with SDZ 242–484 or an EGFR
inhibitor, AG1478. These in vitro studies clearly demonstrate
& 2011 European Molecular Biology Organization
LPA-induced EGFR transactivation in hair follicles
A Inoue et al
A
E
P8
+/+
PA-PLA1α
50 -
TGFα (ng/mg protein)
*
50 -
α-tubulin
50 -
G
2
P2Y5 R = 0.814
PA-PLA1α
1.4
(n = 58)
1.2
1
0.8
0.6
PA-PLA1α
0.4
0.2
α-tubulin
0
Age 0 3 7 10 14 17 19 21 24 28
56
(Postnatal day)
Morph
Cat Tel Ana Tel
Mr (K)
75 -
–/–
75 -
*
F
50 -
H
0.25
+/+
M r (K)
–/–
0.2
IP: p-Tyr
0.15
0.1
*
150 -
*
250 0.05
Input
0
+/+
–/–
Soluble fraction
J
+/+
I
250 -
150 -
Tyrosine-phosphorylated
EGFR
Mr (K)
C
Relative mRNA
expression
D
B
1.2
1
*
0.8
0.6
–/–
0.4
0.2
0
+/+
–/–
IB: EGFR
Figure 4 PA-PLA1a is co-expressed with TACE, TGFa and p-EGFR in hair follicles and EGFR signalling is reduced in PA-PLA1a/ mice. (A–C)
Immunofluorescent staining of PA-PLA1a with TACE (A), TGFa (B) and phosphorylated form of EGFR (p-EGFR) at Tyr1068 (C) in WT dorsal
hair follicles. The arrows indicate co-expression of PA-PLA1a with each protein. Nuclei were stained with DAPI. Magnified views of the boxed
areas are shown in the bottom panels. (D) Immunoblot analysis of PA-PLA1a in dorsal skin lysate. Lysate of skin from P8 WT ( þ / þ ) and PAPLA1a/ (/) mice was subjected to immunoblot using anti-PA-PLA1a antibody. The membrane was stripped and reprobed with anti-atubulin antibody as a loading control. The arrow and asterisk denote PA-PLA1a and non-specific bands, respectively. (E) Hair cycle-dependent
expression of PA-PLA1a in dorsal skin. Skin specimens at different hair cycle stages were analysed by immunoblot as described above. Note that
first hair cycle starts with catagen (P17). Morph, morphogenesis; Cat, catagen (regression); Tel, telogen (resting); Ana, anagen (growth). (F)
qRT–PCR analysis of hair cycle-dependent expression of PA-PLA1a and P2Y5 mRNA in WT dorsal skin. The number of transcripts was
normalized to Gapdh and the maximal value of each gene was set at 1 (n ¼ 3–5). R2, squared correlation coefficient between PA-PLA1a level
and P2Y5 level. (G) Soluble TGFa level in vibrissa hair follicles from WT and PA-PLA1a/ mice. Lysate of vibrissa hair follicles during the
growth phase were ultracentrifuged and the supernatants were subjected to TGFa sandwich ELISA. Amount of soluble TGFa was normalized to
total protein in the same sample determined by the BCA method (mean±s.e.m.; n ¼ 4). (H) p-EGFR level in dorsal skin. Tyrosinephosphorylated proteins were immunoprecipitated from skin lysate during the hair growth phase using anti-phosphotyrosine (p-Tyr)
monoclonal antibody (PY20). The immunoprecipitates (IP) and aliquots of total dorsal skin lysate (Input) were subjected to immunoblot
with anti-EGFR antibody. The arrow and asterisk indicate bands corresponding to p-EGFR and IgG, respectively. (I) Densitometric analysis of
p-EGFR in (H). The IP intensity was normalized to the input intensity in the same sample and the mean value in þ / þ sample was set at 1
(mean±s.e.m.; n ¼ 4). (J) Immunofluorescent staining of p-EGFR at Tyr1068 and K72 in WT and PA-PLA1a/ hair follicles. Nuclei were
stained with DAPI. Scale bars represent 50 mm (A–C, upper panels), 5 mm (A–C, bottom panels) and 20 mm (J). *Po0.05 versus þ / þ .
that LPA transactivates EGFR through ADAMs, which consequently leads to activation of ERK.
We further examined whether P2Y5 and TACE were involved in the process of LPA-induced TGFa release in keratinocytes. Quantitative RT–PCR analysis showed that, among
the six LPA receptors, P2Y5 was most abundantly expressed
in both mouse primary epidermal keratinocytes and human
HaCaT keratinocytes. In addition, the expression patterns of
LPA receptors in the epidermal keratinocytes and HaCaT cells
were nearly identical to those in mouse hair follicles
(Figure 5D). To assess the involvement of P2Y5 and TACE
in TGFa release, we used RNA interference to knockdown
endogenous P2Y5 and TACE in HaCaT cells. Small interfering
& 2011 European Molecular Biology Organization
RNA (siRNA)-mediated knockdown of P2Y5 attenuated the
LPA-induced, but not the TPA-induced, TGFa release, whereas
knockdown of TACE, which was confirmed by immunoblot
analysis (Figure 5E), almost completely inhibited both
LPA-induced and TPA-induced TGFa release (Figure 5F).
Furthermore, the P2Y5 agonists (XY31, XY36 and OMPT)
identified in this study (Supplementary Figure S9A and B)
induced TGFa release from both primary keratinocytes
and HaCaT cells (Figure 5G and H). Notably, a strong
correlation was found between the P2Y5-agonistic activity
(Supplementary Figure S9B) and TGFa-releasing activity
(Figure 5G and H) of these LPA analogues. Taken together,
these data demonstrate that P2Y5 and TACE mediate LPAThe EMBO Journal
VOL 30 | NO 20 | 2011 4253
LPA-induced EGFR transactivation in hair follicles
A Inoue et al
B
**
***
20
**
15
10
5
0
LPA (μM)
TPA
–
–
1
–
10
–
–
+
–
–
10
–
–
+
SDZ 242–484
–
–
–
–
+
+
+
D
35
30
25
20
15
10
5
0
1.2
HaCaT cells
0.8
TACE
0.6
0.4
α-tubulin
0.2
HaCaT cells
0
LPA2
LPA3
LPA4
LPA5
H
TGFα (pg/ml)
30
20
10
P2Y5
10
–
–
+
–
–
1
–
10
–
–
+
1
–
10
–
–
+
20:4
ERK1/2
F
Control siRNA
P2Y5 siRNA
TACE siRNA
***
***
40
30
***
***
**
20
10
0
Vehicle
LPA
TPA
15
10
5
PT
0
T1
M
O
36
31
XY
28
XY
0
PT
M
O
36
T1
31
XY
A
28
XY
XY
LP
Primary keratinocytes
A
0
0
XY
TGFα (pg/ml)
18:0
LPA
1
LPA1
1
–
AG1478
–
–
p-EGFR
18:1
Vehicle
LP
Relative mRNA expression
1.4
–
–
SDZ 242–484
p-ERK1/2
16:0
E
Primary keratinocyte
Vehicle
EGFR
Hair follicle
G
**
*
Pretreatment
LPA (μM)
EGF
**
Control siRNA
25
TGFα (pg/ml)
TGFα (pg/ml)
30
C
TGFα (pg/ml)
**
**
35
TACE siRNA
A
HaCaT cells
Figure 5 LPA induces P2Y5- and TACE-dependent TGFa release and consequent EGFR transactivation in keratinocytes. (A) LPA-induced TGFa
release from primary keratinocytes in a metalloprotease-dependent manner. Keratinocytes were treated with LPA (1-oleoyl, unless otherwise
noted) or a phorbol ester, TPA (100 nM), in the presence or absence of a broad-spectrum metalloprotease inhibitor, SDZ 242–484 (10 mM)
(**Po0.01, ***Po0.001). (B) LPA-induced TGFa release from mouse primary keratinocytes. Keratinocytes were treated with LPA (10 mM)
containing different fatty acids (1-palmitoyl (16:0), 1-oleoyl (18:1) and 1-stearoyl (18:0) and 1-arachidonoyl (20:4)). TGFa in the conditioned
media was measured by sandwich ELISA (*Po0.05, **Po0.01 versus vehicle treatment). (C) Immunoblot analyses of EGFR and ERK
phosphorylation in primary keratinocytes. Cells were treated with LPA or EGF (100 ng/ml) for 5 min (p-EGFR) or 15 min (p-ERK) in the
presence or absence of SDZ 242–484 (10 mM) or an EGFR inhibitor, AG1478 (1 mM). (D) Expressions of LPA receptors in P4 dorsal hair follicles,
primary keratinocyte and human HaCaT keratinocytes (n ¼ 4). The number of transcripts was normalized to Gapdh and the mean value of
P2Y5 expression level was set at 1 (n ¼ 3–4). (E) Immunoblot analysis of siRNA-mediated knockdown of TACE in HaCaT cells. (F) TGFa release
from siRNA-transfected HaCaT cells. Control, P2Y5 or TACE-knockdown cells were treated with LPA (10 mM) or TPA (100 nM) and TGFa release
was measured (**Po0.01, ***Po0.001). Note that knockdown efficiency (mRNA reduction level quantified by qRT–PCR) of TACE siRNA and
P2Y5 siRNA was 76±4.5% (mean±s.d., n ¼ 3) and 67±4.8%, respectively. (G, H) LPA analogues (10 mM) were tested for TGFa-releasing
activities from primary keratinocytes (D) and HaCaT cells (E). The TGFa concentration in vehicle-treated cells was set as the baseline. Note that
TGFa release was potently induced by P2Y5 agonists (XY31, XY36 and OMPT), but not by LPA analogues that lack P2Y5-agonistic activity
(XY28 and T10). The structures and agonistic activities of these LPA analogues at LPA receptors are shown in Supplementary data. (A, B, F, G,
H) Data are representative of three independent experiments and show the mean±s.d. of at least triplicate cultures.
induced TGFa release and the consequent EGFR transactivation in keratinocytes.
Reconstitution of LPA-induced TGFa release in
HEK293 cells
As a further test of whether P2Y5 has a role in TGFa release,
we reconstituted a cell-based system that mimics ectodomain
shedding of TGFa by utilizing an alkaline phosphatase (AP)tagged TGFa (AP-TGFa) (Tokumaru et al, 2000), which we
refer to as an AP-TGFa shedding assay (Figure 6A). In this
system, AP-TGFa release into the conditioned media can be
quantitatively measured by a colorimetric reaction using
p-nitrophenylphosphate (p-NPP). When HEK293 cells transiently expressing AP-TGFa and P2Y5 (either mouse P2Y5 or
human P2Y5) were treated with LPA, AP activity was dose
4254 The EMBO Journal VOL 30 | NO 20 | 2011
dependently increased in the conditioned media (Figure 6B),
showing that P2Y5 mediated LPA-induced AP-TGFa release.
P2Y5-expressing cells and mock-transfected (control) cells
were equally sensitive to TPA stimulation (Figure 6C),
indicating that expression of P2Y5 did not affect PKC-induced
AP-TGFa release. Importantly, no significant AP-TGFa release
was induced from P2Y5-expressing cells by the other six
lysophospholipids (Figure 6C; Supplementary Figure S8A),
confirming that P2Y5 is an LPA-specific receptor. We noticed
that a small amount of AP-TGFa was also released from
control cells, possibly via endogenously expressed LPA
receptors in HEK293 cells (Supplementary Figure S7).
Indeed, siRNA-mediated knockdown of P2Y5 endogenously
expressed in HEK293 cells attenuated LPA-induced AP-TGFa
release by 45%, but did not attenuate TPA-induced AP-TGFa
& 2011 European Molecular Biology Organization
LPA-induced EGFR transactivation in hair follicles
A Inoue et al
AP activity on cell surface
AP-TGFα
AP activity in
conditioned media (%)
Total AP activity (100%)
A
AP activity in conditioned media
Lysophospholipid
Transfer conditioned media
add p-NPP solution
Proteinase
HEK293 cell
P2Y5
C
mP2Y5
Control
20
AP activity release (%)
AP activity release (%)
hP2Y5
** **
**
15
**
10
** **
*
5
**
**
0
0
10
100
LPA (nM)
1000
mP2Y5
LPA
LPC
LPE
***
AP activity
release (%)
LPA
α-tubulin
P2Y5
HEK293 cells
TPA
H
AP activity
release (%)
25
NS
20
15
10
5
0
Inhibitor Vehicle NF449
NS
PTX
YM Y27632 U73122
LPA
**
Ro
NS
45
40
**
10
5
0
LPA
TPA
***
***
***
Control
P2Y5 Control
LPA
Control vector
NS
***
Control siRNA
P2Y5 siRNA
P2Y5
TPA
J
P2Y5 vector
NS
5
NS
I
Control vector
Treatment
TPA
***
***
Treatment
AP activity
release (%)
Treatment
50
40
30
20
10
0
Vector
TACE
S1P
LPS
Control siRNA
TACE siRNA
AP activity
release (%)
Control
siRNA
***
LPI
G
NS
50
***
40
***
30
20
***
10
0
Vector Control P2Y5 Control
LPG
AP activity
release (%)
10
Control
**
**
F
Vehicle
SDZ 242–484
hP2Y5
TACE
siRNA
E
D
45
40
35
20
15
10
5
0
15
0
Treatment Vehicle Lysophospholipid
pro-AP-TGFα
AP activity release (%)
B
AP-TGFα
pro-AP-TGFα
TPA
p - Nitrophenol
p - NPP
Proteinase
Colorimetric reaction (OD405)
P2Y5
p - Nitrophenol
p - NPP
20
20
P2Y5 vector
AP-TGFα
LPA
TACE
*
15
**
P2Y5
**
10
G12
G13
5
0
Vector Control DN G12 DN G13 DN RhoA
Treatment
RhoA
pro-AP-TGFα
PKC
ROCK
LPA
Figure 6 LPA induces P2Y5- and TACE-dependent TGFa release via a G12/13–RhoA–ROCK pathway in HEK293 cells. (A) Scheme of AP-tagged
(AP-TGFa) shedding assay. HEK293 cells transiently expressing AP-TGFa and P2Y5 were treated with a compound for 1 h. Amount of AP-TGFa
release was quantified by measuring AP activities in both conditioned media and cell surface using a colorimetric reaction of AP substrate,
p-NPP. Note that protease that cleaves AP-TGFa is endogenously expressed in HEK293 cells. AP activity detected in vehicle treatment was set as
the baseline. (B) LPA induces AP-TGFa release from P2Y5-expressing cells. HEK293 cells that were transfected with mouse P2Y5 (mP2Y5),
human P2Y5 (hP2Y5) or control vector were stimulated with 3–1000 nM of LPA (n ¼ 4; *Po0.05, **Po0.01 versus control cells). (C) Same as
(B), but treated with various lysophospholipids (1 mM) and TPA (100 nM). Note that five lysophospholipids other than LPA did not induce
P2Y5-dependent AP-TGFa release. Also note that transient expression of P2Y5 did not affect TPA (100 nM)-induced AP-TGFa release.
(D) siRNA-mediated knockdown of P2Y5 endogenously expressing in HEK293 cells attenuated LPA-induced, but not TPA-induced, AP-TGFa
release. Note that knockdown efficiency (mRNA reduction level quantified by qRT–PCR) with P2Y5 siRNA was 85±1.8% (mean±s.d., n ¼ 3).
(E–G) SDZ 242–484 (a metalloprotease inhibitor; 1 mM) (E) or siRNA-mediated knockdown of TACE (F, G) inhibited both LPA (1 mM)-induced
and TPA (100 nM)-induced AP-TGFa release. Note that knockdown efficiency with TACE siRNA was 90±0.7% (mean±s.d., n ¼ 3). (H) LPA/
P2Y5-induced AP-TGFa release was blocked by Y27632 (a ROCK inhibitor; 10 mM; 30 min pretreatment, unless otherwise noted) and Ro31-8425
(Ro; a pan-PKC inhibitor; 10 mM), but not by NF449 (a Gs inhibitor, 100 mM), PTX (a Gi inhibitor; 100 ng/ml; 16 h), YM254890 (YM; a Gq
inhibitor; 10 mM) and U73122 (a PLCb inhibitor; 3 mM). (I) LPA/P2Y5-induced AP-TGFa release was blocked by co-transfection of DN forms of G
proteins including G12, G13 (heterotrimeric Ga proteins) and RhoA (small G protein). Concentration of LPA was 1 mM (H, I). Bars and error
bars in (C–E, G–I) represent mean values and s.d., respectively (n ¼ 3–4; *Po0.05, **Po0.01, ***Po0.001; NS, not significant difference). (J)
Scheme of signal transduction leading to TGFa release in HEK293 cells. Upon binding of LPA, P2Y5 activates G12/13–RhoA–ROCK signalling,
which leads to TACE-dependent ectodomain shedding of pro-TGFa, partially through a PKC pathway, and results in TGFa release.
release (Figure 6D). Similar to TGFa release from keratinocytes, both LPA-induced and TPA-induced AP-TGFa release
from HEK293 cells was almost completely suppressed by
knockdown of TACE and pretreatment with a broad metalloprotease inhibitor, SDZ 242–484 (Figure 6E–G). Using the
reconstituted model, we further examined signal transduction
& 2011 European Molecular Biology Organization
of P2Y5 leading to AP-TGFa release in HEK293 cells. Among
four inhibitors that specifically block four distinct subclasses
of heterotrimeric Ga proteins (Gs, Gi, Gq and G12/13), only
Y27632 (a Rho kinase (ROCK) inhibitor; G12/13 pathway)
inhibited LPA/P2Y5-induced AP-TGFa release, whereas
NF449 (a Gs inhibitor), Pertussis Toxin (PTX; a Gi inhibitor)
The EMBO Journal
VOL 30 | NO 20 | 2011 4255
LPA-induced EGFR transactivation in hair follicles
A Inoue et al
manner via hydrolysis of PA on the plasma membrane and
production of LPA.
and YM254890 (a Gq inhibitor) did not have the inhibitory
effect (Figure 6H). In addition, Ro31-8425 (a pan-PKC
inhibitor), but not U73122 (a PLCb inhibitor), blocked LPA/
P2Y5-induced AP-TGFa release. Co-transfection of dominantnegative (DN) forms of G proteins including G12, G13 and
RhoA also inhibited LPA/P2Y5-induced AP-TGFa release
(Figure 6I), confirming the involvement of G12/13 signalling.
Thus, we concluded that LPA/P2Y5-induced G12/13 signalling underlies TACE-dependent TGFa release (Figure 6J).
Discussion
Genetic studies of human hair disorders have suggested that
LPA signalling has a crucial role in hair follicle development.
However, the underlying molecular mechanism, even the
involvement of LPA itself, has until now been unclear. The
data presented here show that LPA generated by an LPAproducing enzyme, PA-PLA1a, regulates hair follicle formation through a mechanism that involves TGFa release
and EGFR transactivation via the most recently identified
LPA receptor, P2Y5 (Figure 8). This model is supported by the
following observations: (i) PA-PLA1a/ mice exhibited a
wavy hair phenotype that resembles the hair phenotype of
mutant mice defective in TGFa-related genes (TACE, TGFa
and EGFR) (Luetteke et al, 1993, 1994; Mann et al, 1993;
Peschon et al, 1998; Hassemer et al, 2010); (ii) PA-PLA1a,
TACE, TGFa and p-EGFR were co-expressed in the IRS and,
PA-PLA1a and P2Y5 showed similar hair cycle-dependent
expression; (iii) the activation states of TGFa and EGFR
were decreased in PA-PLA1a/ mice; (iv) LPA species with
unsaturated fatty acids, which were found to be potent
agonists for P2Y5, were selectively lowered in PA-PLA1a/
mice; (v) LPA induced TGFa release and EGFR transactivation
via P2Y5 and TACE in keratinocytes; and (vi) LPA and PAPLA1a induced P2Y5-mediated TGFa release in a TACE-dependent manner in reconstituted HEK293 cells. In vitro
studies have demonstrated that LPA is an initiator of EGFR
transactivation in a variety of cells such as corneal epithelial
cells and lung epithelial cells (Prenzel et al, 1999; Zhao et al,
2006; Xu et al, 2007). This study is the first to demonstrate
the in vivo significance of LPA-induced EGFR transactivation.
Moreover, GPCR-induced EGFR transactivation has been
PA-PLA1a initiates P2Y5-mediated TGFa release in
HEK293 cells
We finally asked whether PA-PLA1a itself is capable of inducing P2Y5-mediated AP-TGFa release. Co-expression of P2Y5
and PA-PLA1a, but not a catalytically inactive PA-PLA1a
mutant (S154A) (Sonoda et al, 2002), dramatically enhanced
AP-TGFa release (Figure 7A). A small but significant APTGFa release was observed when PA-PLA1a vector was
introduced alone (Figure 7A, control þ WT), probably via
endogenously expressed LPA receptors in HEK293 cells
(Supplementary Figure S7), as was observed in LPA-stimulated mock-transfected cells (Figure 6B and D). Next, we
tested whether PA-PLA1a was capable of activating P2Y5 in a
juxtacrine manner. When PA-PLA1a-expressing cells and
P2Y5-expressing cells were prepared separately and mixed
together, significant AP-TGFa release from P2Y5-expressing
cells was observed (Figure 7B). The AP-TGFa release was
also dependent on the catalytic activity of PA-PLA1a and the
transfection of the P2Y5 vector. The PA-PLA1a-initiated APTGFa release from P2Y5-expressing cells was further elevated
upon treatment with bacterial phospholipase D (PLD)
(Figure 7C), which was previously shown to generate PA on
the outer leaflet of plasma membrane (Sonoda et al, 2002;
Aoki, 2004). These data clearly show that PA-PLA1a is capable of activating P2Y5 in an autocrine and/or juxtacrine
A
B
C
pro-AP-TGFα
PA-PLA1α
pro-AP-TGFα
PA-PLA1α
P2Y5
pro-AP-TGFα
PA-PLA1α
P2Y5
P2Y5
PA
PA
AP activity release (%)
6
#
**
**
**
4
*
2
P2Y5 + WT
P2Y5 + S154A
Control + WT
Control + S154A
##
##
**
**
**
0
AP activity release (%)
P2Y5 + WT
P2Y5 + S154A
Control + WT
Control + S154A
8
8
6
**
**
**
4
*
2
AP activity release (%)
PLD
P2Y5 + WT
P2Y5 + S154A
Control + WT
Control + S154A
25
20
**
*
15
10
5
0
0
0
10
100
1000
10 000
PA-PLA1α-expression vector (pg per well)
0
1
10
100
3
PA-PLA1α-expressing cells (×10 cells per well)
0
1
10
100
1000
Actinomadura PLD (mU/ml)
Figure 7 PA-PLA1a induces P2Y5-dependent TGFa release. PA-PLA1a-mediated activation of P2Y5 in autocrine (A) and paracrine (B, C)
manners. (A) Spontaneous AP-TGFa release from HEK293 cells expressing catalytically active PA-PLA1a (WT), P2Y5 and AP-TGFa. Note that
AP-TGFa release was not observed in cells expressing catalytically inactive PA-PLA1a mutant harbouring a serine-to-alanine substitution at
residue 154 (S154A). Also note that AP-TGFa release was significantly reduced when P2Y5 vector was replaced with an empty vector (control)
(n ¼ 4; *Po0.05, **Po0.01 versus control vector-transfected cells; #Po0.05, ##Po0.01 versus S154A-expressing cells). (B) PA-PLA1a-induced
paracrine activation of P2Y5. P2Y5- and AP-TGFa-expressing cells were mixed with PA-PLA1a-expressing cells that were separately prepared
(n ¼ 4; *Po0.05, **Po0.01 versus control vector-transfected cells and S154A-expressing cells). (C) Exogenous phospholipase D (PLD) potently
enhanced the PA-PLA1a-mediated AP-TGFa release. Same as B (2 103 PA-PLA1a-expressing cells per well), but in the presence of bacterial
PLD. AP activity in the absence of PA-PLA1a-expressing cells and PLD treatment was used as the baseline (n ¼ 4; *Po0.05, **Po0.01 versus
control vector-transfected cells and S154A-expressing cells).
4256 The EMBO Journal VOL 30 | NO 20 | 2011
& 2011 European Molecular Biology Organization
LPA-induced EGFR transactivation in hair follicles
A Inoue et al
PA-PLA1α
PA
2-acyl-LPA
PA-PLA1α
TACE
PA
P2Y5
TGFα
pro-TGFα
EGFR
Inner root sheath
Hair follicle development
Figure 8 Proposed model for PA-PLA1a- and P2Y5-mediated hair
follicle development. PA-PLA1a is expressed and secreted in the
developing IRS of hair follicles and produces 2-acyl-LPA from PA on
the outer leaflet of the plasma membrane by hydrolyzing the acyl
chain at the sn-1 position. The resulting 2-acyl-LPA activates P2Y5
in a paracrine and/or autocrine manner, eliciting TACE-dependent
shedding of membrane-bound TGFa (pro-TGFa). Soluble TGFa
binds to EGFR in the IRS of hair follicles. Activated/phosphorylated
EGFR induces development of the IRS, which is required for proper
formation of the hair shaft. Genetic deletions of these molecules
(PA-PLA1a in this study and Kazantseva et al (2006), P2Y5
(Pasternack et al, 2008; Shimomura et al, 2008), TACE (Peschon
et al, 1998; Hassemer et al, 2010), TGFa (Luetteke et al, 1993; Mann
et al, 1993), EGFR (Luetteke et al, 1994)) result in aberrant hair
formation in mice and/or humans.
implicated in several pathophysiological conditions such as
renal lesions (Lautrette et al, 2005), wound healing (mediated
by AT1) (Yahata et al, 2006), cardiac hypertrophy (AT1 and
b1) (Zhai et al, 2006; Noma et al, 2007) and regulation of
blood pressure (endothelin-1 receptors) (Chansel et al, 2006).
However, these previous studies examined disease models
that were, in most cases, induced by exogenously administered GPCR agonists. Thus, our data provide the first
evidence of the physiological role of GPCR-induced EGFR
transactivation in vivo.
Recent experiments with mouse embryonic fibroblasts
suggest that LPA signalling shares a common pathway with
EGFR signalling (Stortelers et al, 2008). Because LPA-induced
EGFR transactivation can be mediated by not only P2Y5
but also by other LPA receptors such as LPA1 and LPA3
(Supplementary Figure S10B and C), it is reasonable to
assume that LPA-producing enzymes and LPA receptors
may cooperatively function in EGFR transactivation, thereby
affecting other EGFR-involved physiological and pathophysiological conditions such as wound healing, development of
heart and bone and progression of cancer (Sibilia and
Wagner, 1995; Threadgill et al, 1995).
Activity of ADAMs is regulated by several kinases mainly
through phosphorylation of a cytoplasmic region of these
proteases in some systems (Mezyk et al, 2003; Edwards et al,
2008). These kinases are activated downstream of both
receptor tyrosine kinases and GPCRs. Although some
G proteins such as Gi and Gq are implicated in activation of
ADAMs and consequent EGFR ligand release (Ohtsu et al,
2006; Uchiyama et al, 2009), little is known about G proteins
involved in TACE activation. Our data establish a G12/
13–RhoA–ROCK pathway, induced by LPA/P2Y5, as a novel
regulator of TACE activation (Figure 6H–J). It should be noted
that, consistent with our observation, Yanagida et al (2009)
also showed that P2Y5 coupled with G13 and induced Rho
& 2011 European Molecular Biology Organization
signalling pathway. In general, G12/13 signalling, compared
with Gs, Gi and Gq signalling, is considered an experimentally difficult pathway to detect in conventional GPCR assays
such as cAMP, Ca2 þ mobilization and promoter-driven
luciferase (Siehler, 2009). Our results suggest that AP-TGFa
release may be a useful readout, which may uncover
previously unidentified or unappreciated GPCRs coupled
with G12/13.
So far physiological importance of 2-acyl-LPA has not been
elucidated. The present study demonstrated for the first time
that 2-acyl-LPA is present in hair follicles and has a critical
and specific role in hair follicle formation. Using the LC-MS/
MS method established in this study, we demonstrated that
the levels of 2-acyl-LPA species with unsaturated fatty acids
were reduced in PA-PLA1a/ hair follicles (Figure 2A–C),
which demonstrates that PA-PLA1a produces 2-acyl-LPA
in vivo. Thus, it seems likely that 2-acyl-LPA species with
unsaturated fatty acids act as ligands of P2Y5 in hair follicles.
In agreement with this, we found that P2Y5 was more
strongly activated by LPA species with unsaturated fatty
acids than by LPA species with saturated fatty acids
(Supplementary Figure S8C). We previously showed that
2-acyl-LPA with unsaturated fatty acids preferentially
activated LPA3 (Bandoh et al, 2000), which has a critical
role in embryo implantation in the uterus (Ye et al, 2005).
Together with these previous studies on LPA3, the present
study on PA-PLA1a and P2Y5 underscores the importance of
2-acyl-LPA in regulating physiological events.
It is likely that 2-acyl-LPA generated on a membrane by
PA-PLA1a immediately acts on P2Y5 in an autocrine and/or
juxtacrine manner. This model is supported by the following
observations: (i) PA-PLA1a is a secreted membrane-bound
enzyme (Sonoda et al, 2002) and is concentrated on the
plasma membrane (Hiramatsu et al, 2003); (ii) PA-PLA1
a-expressing cells (Figure 7A and B), but not the conditioned
media from the cells (data not shown), efficiently induced
P2Y5-mediated AP-TGFa release in an autocrine or juxtacrine
manner; and (iii) both PA-PLA1a (Figure 3G) and P2Y5
(Shimomura et al, 2008) are expressed in the IRS of the
hair follicles. This model is also supported by the fact that 2acyl-LPA is generally unstable due to a reaction known as
spontaneous acyl migration. Furthermore, the fact that
PA-PLA1a directly activated P2Y5 indicates that PA is located
in the outer leaflet of the plasma membrane and, more
importantly, PA transporter/scramblase exists, similar to a
recently identified PS scamblase (Suzuki et al, 2010), because
PA is intracellularly synthesized.
PA-PLA1a belongs to the lipase family, which includes
pancreatic lipase and lipoprotein lipase. PA-PLA1a, together
with PA-PLA1b and PS-specific PLA1 (PS-PLA1), form a
subfamily within the lipase family. Unlike other lipases,
these PLA1s exhibit strict substrate specificity to either PA
(PA-PLA1a and b) (Sonoda et al, 2002; Hiramatsu et al, 2003)
or PS (PS-PLA1) (Sato et al, 1997; Aoki et al, 2007). Our
recent studies (paper in preparation) indicate that PS-PLA1
produces in vivo LPS, another lysophospholipid mediator
with a variety of biological functions (Aoki et al, 2007;
Makide et al, 2009). It is noteworthy that a unique feature
of the products of these enzymes is that they have a fatty acid
attached at the sn-2 position of the glycerol backbone. Our
results show that 2-acyl-LPA is a potent ligand of P2Y5. Thus,
from biochemical and structural points of view, we speculate
The EMBO Journal
VOL 30 | NO 20 | 2011 4257
LPA-induced EGFR transactivation in hair follicles
A Inoue et al
that an ancestral member of the lipase family that had a
specific role in production of lysophospholipid mediators,
such as LPA and LPS, emerged and evolved through gene
duplication, which eventually formed the PLA1 subfamily.
Materials and methods
Reagents, antibodies and plasmids
See Supplementary data.
Animals
Mice were maintained according to the Guidelines for Animal
Experimentation of Tohoku University and the protocol was approved
by the Institutional Animal Care and Use Committee at Tohoku
University (No. 21-Pharm-Animal-21). Generation of PA-PLA1a/
mice is described in Supplementary data. In all experiments using
homozygous mice, littermates were used for a control.
Histology and in situ hybridization analyses
See Supplementary data.
Immunofluorescent staining
Paraformaldehyde-fixed tissues were embedded in OTC compound
(Sakura Finetek) or in paraffin wax. For paraffin sections, antigen
retrieval was performed using 10 mM citrate buffer (pH 7.0) at
1201C for 10 min. PA-PLA1a was detected using biotinylated anti-PAPLA1a monoclonal antibody (clone mask1), followed by ABC Elite
kit (Vector Laboratories) and TSA Alexa Fluor 488 kit (Invitrogen).
Except for anti-PA-PLA1a monoclonal antibody, primary antibodies
were visualized with secondary antibodies conjugated to Alexa
Fluor 488, 568, 594 and 647. Nuclei were counterstained with 40 ,
6-diamidino-2-phenylindole (DAPI). Fluorescent images were
captured with a Zeiss LSM700 confocal microscope (Carl Zeiss).
Quantitative RT–PCR analysis
See Supplementary data. The primers used in the qRT–PCR analysis
are listed in Supplementary data.
Isolation of epidermal keratinocytes and dorsal hair follicles
See Supplementary data.
Cell culture and transfection
See Supplementary data.
Quantification of endogenous TGFa release from
keratinocytes
The media of primary mouse epidermal keratinocytes and HaCaT
cells were replaced with 250 ml of supplement-free K-SFM and
serum-free DMEM, respectively, on the day of cell stimulation. One
hour later, primary keratinocytes were treated with various
compounds for 2 h. For metalloprotease inhibitor treatment, cells
were pretreated with 10 mM SDZ 242–484 for 30 min. TGFa levels
in conditioned media were measured with a TGFa sandwich ELISA
(human TGFa DuoSet Kit, R&D Systems) according to the
manufacturer’s instructions. The TGFa sandwich ELISA kit can
recognize murine TGFa.
Immunoblot and immunoprecipitation
See Supplementary data.
Determination of soluble TGFa in hair follicles
Vibrissa hair follicles were surgically isolated from P15 C57BL6/J mice
by removing the collagen capsules in ice-cold PBS containing 1 mM
EDTA and 10 mM SDZ 242–484 (see also Supplementary Figure S4A).
Ten vibrissa hair follicles per a single mouse were extracted with a
MS-100R beads cell disrupter in lysis buffer (25 mM HEPES (pH 7.3),
250 mM sucrose, 10 mM MgCl2, 1 mM EDTA, 10 mg/ml PMSF, 20 mg/ml
leupeptin and 10 mM SDZ 242–484). Lysate was centrifuged at 1000 g
for 10 min. The supernatant was centrifuged at 200 000 g for 30 min.
Protein concentration in the resulting supernatant (soluble fraction)
was measured with a Micro BCA Protein Assay Kit (Pierce) and an
equal amount of protein was used for TGFa sandwich ELISA.
Quantification of lysophospholipids by LC-MS/MS
See Supplementary data.
4258 The EMBO Journal VOL 30 | NO 20 | 2011
AP-TGFa shedding assay
AP-TGFa release was assayed by the previously described method
(Tokumaru et al, 2000) with several modifications. HEK293 cells
transiently expressing AP-TGFa with or without P2Y5 were
detached with 0.05% Trypsin/0.52 mM EDTA, resuspended in
Hank’s balanced salt solution (HBSS) containing 5 mM HEPES
(pH 7.4), and seeded in 90 ml at 2 104 cells per well in a 96-well
plate. After 30 min, cells were treated with 10 ml of 10 concentration of compounds for 1 h. After transferring 80 ml of conditioned
media in each well to a new 96-well plate, 80 ml of p-NPP solution
(10 mM p-NPP, 20 mM Tris–HCl (pH 9.5), 20 mM NaCl, 5 mM
MgCl2) was added to both conditioned media and cells. OD405 was
measured before and after 1 h incubation at 371C with a VersaMax
microplate reader (Molecular Devices). AP activity release was
calculated from the increase of OD405 during 1 h incubation in the
conditioned media (DODmedia) and the cell (DODcell) as follows
(also see Figure 7A): AP activity in the conditioned media (APmedia)
(%) is defined as the ratio of DODmedia to total DOD values
(DODmedia plus DODcell); AP activity release (%) is determined by
subtracting basal APmedia from APmedia in the presence of a
compound where basal APmedia is defined as APmedia in the absence
of a compound. Note that basal APmedia varied from 5 to 12%,
depending on assay conditions.
For autocrine PA-PLA1a-initiated AP-TGFa release, HEK293 cells
were transfected with combinations of PA-PLA1a, P2Y5 and APTGFa vectors in 100 ml of Opti-MEM (GIBCO) at 2 104 cells per
each 96 well. Twenty-four hours after transfection, AP activity
release was determined as described above. For paracrine
PA-PLA1a-initiated AP-TGFa release, P2Y5 and AP-TGFa-expressing
cells were prepared and seeded in a 96-well plate as described
above. PA-PLA1a-expressing HEK293 cells were resuspended
in HBSS and added to P2Y5 and AP-TGFa-expressing cells. After
1 h of incubation at 371C, AP activity release was determined.
For exogenous PLD treatment, 2 104 of P2Y5 and AP-TGFaexpressing cells and 2 104 of PA-PLA1a-expressing cells were
mixed and incubated for 1 h in the presence or absence of bacterial
PLD. AP activity release was determined. Catalytically inactive PAPLA1a mutant (S154A) and empty vector were used as negative
controls for PA-PLA1a and P2Y5, respectively.
Statistics
Columns and error bars represent means and standard deviations of
independent experiments, respectively, unless otherwise indicated.
Student’s t-test was used to determine the significance of
differences between groups. One-way ANOVA with Bonferroni’s
posttest analyses of multiple groups was employed for analyses of
multiple groups. For all statistical tests, the 0.05 level of confidence
was accepted for statistical significance.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We thank Dr Hitoshi Okochi and Dr Aki Osada (National Center for
Global Health and Medicine, Japan) for technical advice on hair
follicle analyses. JA was supported by grants from the National
Institute of Biomedical Innovation, the National Project on Protein
Structural and Functional Analyses from the Ministry of Education,
Science, Sports and Culture of Japan (MEXT) and PRESTO from Japan
Science and Technology Corporation. IA was a research fellow of the
Japan Society for the Promotion of Science (DC1 1811607). IA was
supported by a Grant-in-Aid for Young Scientists (B) (KAKENHI
21790058), Takeda Science Foundation and The Naito Science &
Engineering Foundation. GDP thanks the Human Frontier of Science
Program and the NIH (NS29632) for financial support.
Author contributions: AI, NA and JI performed the experiments
and analysed the data. GDP developed LPA analogues. AI and JA
prepared the paper. HA and JA supervised the work.
Conflict of interest
The authors declare that they have no conflict of interest.
& 2011 European Molecular Biology Organization
LPA-induced EGFR transactivation in hair follicles
A Inoue et al
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