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Bone morphogenetic proteins differentially regulatepigmentation in human skin cells
Suman K. Singh, Waqas A. Abbas and Desmond J. Tobin*Centre for Skin Sciences, School of Life Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK
*Author for correspondence ([email protected])
Accepted 25 April 2012Journal of Cell Science 125, 4306–4319� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.102038
SummaryBone morphogenetic proteins (BMPs) are a large family of multi-functional secreted signalling molecules. Previously BMP2/4 wereshown to inhibit skin pigmentation by downregulating tyrosinase expression and activity in epidermal melanocytes. However, a possible
role for other BMP family members and their antagonists in melanogenesis has not yet been explored. In this study we show that BMP4and BMP6, from two different BMP subclasses, and their antagonists noggin and sclerostin were variably expressed in melanocytes andkeratinocytes in human skin. We further examined their involvement in melanogenesis and melanin transfer using fully matched primary
cultures of adult human melanocytes and keratinocytes. BMP6 markedly stimulated melanogenesis by upregulating tyrosinaseexpression and activity, and also stimulated the formation of filopodia and Myosin-X expression in melanocytes, which was associatedwith increased melanosome transfer from melanocytes to keratinocytes. BMP4, by contrast, inhibited melanin synthesis and transfer to
below baseline levels. These findings were confirmed using siRNA knockdown of BMP receptors BMPR1A/1B or of Myosin-X, as wellas by incubating cells with the antagonists noggin and sclerostin. While BMP6 was found to use the p38MAPK pathway to regulatemelanogenesis in human melanocytes independently of the Smad pathway, p38MAPK, PI3-K and Smad pathways were all involved in
BMP6-mediated melanin transfer. This suggests that pigment formation may be regulated independently of pigment transfer. These datareveal a complex involvement of regulation of different members of the BMP family, their antagonists and inhibitory Smads, inmelanocytes behaviour.
Key words: Pigmentation, Melanin transfer, UV radiation, Noggin, Sclerostin
IntroductionSkin pigmentation, a critical phenotypic adaptation for ultra-violetradiation (UVR)-drenched terrestrial life, is dependent on the
activity of melanocytes (MC). This subpopulation of neural crest-derived cells migrates during embryogenesis to the epidermis and
hair follicles and subsequently synthesize and distribute melanin tosurrounding keratinocytes (KC) (Van Den Bossche et al., 2006;
Tobin, 2008). Adequate pigmentation of skin is dependent upon
successful transport and transfer of a unique membrane-bound andlysosome-related organelle, the melanosome, from MC to KC
(Van Den Bossche et al., 2006). Such whole organelle donationfrom one cell to another heterotypic cell is unique to the
melanocyte–keratinocyte unit. After transfer, melanin is
generally transported to the apical surface of the KC nucleus,which it protects against UVR damage. The complex process of
melanosome biogenesis, trafficking and intracellular transport hasbeen extensively studied (Vancoillie et al., 2000; Byers et al.,
2000). However, just how melanin is transferred to neighbouringKCs has remained stubbornly enigmatic (Van Den Bossche et al.,
2006). Several hypotheses have been proposed including:
(i) cytophagocytosis of MC dendrite tips (Seiberg et al., 2000),(ii) exocytosis of melanosomes and subsequent uptake via
phagocytosis into KC (Marks and Seabra, 2001; Virador et al.,2002) and (iii) filopodia-mediated melanosome transfer (Singh
et al., 2010; Singh et al., 2008; Beaumont et al., 2011; Zhang et al.,
2004; Scott et al., 2002). The role of filopodia in the transfer ofmelanosomes to keratinocytes was originally proposed by Scott
and colleagues (Scott et al., 2002), and we extended this further in
our recent report that proposed a ‘filopodial-phagocytosis model’for melanin transfer (Singh et al., 2010). However, the underlying
regulatory and signalling pathways involved remain poorly
defined, and this is the subject of the current study.
Bone morphogenetic proteins (BMPs) are powerful regulatorsof skin development where they play important roles in the
control of epidermal homeostasis, hair follicle growth and
pigmentation (Botchkarev et al., 1999; Botchkarev et al., 2001;Botchkarev, 2003; Botchkarev and Sharov, 2004; Sharov et al.,
2005; Sharov et al., 2006). BMP signalling operates at multiplelevels depending on BMP type, presence of antagonist(s),
development stage of the target tissue, and the target cell
receptor type (Botchkarev, 2003). Once secreted, binding of theBMP to at least one type-I and one type-II receptor is necessary
for activation of the BMP signal (ten Dijke et al., 1996).Depending on cell type BMPs exert their effects by binding to
these BMP receptors to transduce signals to the nucleus byrecruiting Smad1/5/8 transcription regulators or components of
the MAPK pathway or PI3-K (Miyazono et al., 2005; Osyczka
and Leboy, 2005; Herpin and Cunningham, 2007). BMPreceptors BMPR1A (ALK3), BMPR1B (ALK6) and BMPR-II
have been shown to assemble either as preformed hetero-oligomeric (BMPR-II/ALK3 and BMPR-II/ALK6) or homo-
oligomeric (BMPR-II/BMPR-II, ALK3/ALK3, ALK6/ALK6 and
ALK3/ALK6) complexes in the absence of ligand (Gilboa et al.,2000). Thus, a ligand such as BMP6 has two options to initiate
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signal transduction. It can bind to the high-affinity receptor ALK3
or ALK6 and then recruit BMPR-II into a hetero-oligomeric
complex, leading to activation of the p38 MAPK pathway (Nohe
et al., 2002). Alternatively, it can bind simultaneously to the
preformed hetero-oligomeric complexes consisting of at least one
type-I and one type-II receptor, and then activating the Smad
signalling pathway; the so-called canonical BMP/Smad pathway
(Nohe et al., 2004). Furthermore, extracellular antagonists are
crucial in controlling BMP signalling outcomes (Balemans and
Van Hul, 2002).
To date, approximately 20 unique BMP ligands have been
identified (Miyazono et al., 2010). BMP4 and BMP6 belong to
the BMP2/4 subclass and BMP5/6/7 subclass respectively
(Miyazono et al., 2010). It has recently been shown that BMP4
down-regulates melanogenesis by microphthalmia-associated
transcription factor (MITF) degradation, thereby reducing the
level of tyrosinase expression via preferential utilization of
MAPK/ERK pathways (Yaar et al., 2006; Park et al., 2009a; Park
et al., 2009b). However, there is no data on the influence of
BMP4 on other aspects of melanocyte behaviour (incl. melanin
transfer). We know nothing of the mechanism through which
BMP6 affects pigmentation, either via melanin synthesis (i.e.
melanogenesis) or melanosome transfer from donor MC to
recipient KC.
Here we address several fundamental questions relating to how
BMP regulates skin pigmentation. (1) Are BMP4 and BMP6, and
their natural antagonists noggin and sclerostin respectively,
expressed in normal human adult skin cells? (2) Is the
expression of BMP4 and BMP6, their BMP receptors and
antagonists regulated by UVR? (3) Do BMP6 and BMP4 have
similar regulatory effects on melanogenesis? (4) Do BMP4 and
BMP6 differentially control the process of melanin transfer. (5)
Which signalling pathway(s) is/are used by BMP6 to regulate
melanogenesis and melanin transfer in human epidermal skin cells,
and are these differentially regulated processes?
Here we provide evidence that BMP6 markedly stimulates
MC melanogenesis and melanin transfer from MC to KC (in
contradistinction to BMP4), and we define distinctive signalling
pathways involved in these unexpected opposing effects.
ResultsNormal adult human skin expresses BMP6 and BMP4, and
their respective receptors and antagonists
Strong expression of BMP6 and BMP4, and their respective
antagonists (sclerostin and noggin) and receptors (ALK3, ALK6)
was detected in epidermal keratinocytes (KC) and in gp100-
positive epidermal melanocytes (MC) (Fig. 1A–F), indicating
that adult skin is very likely a BMP6-responsive tissue.
The expression of BMP6 and its receptors is upregulated
by UVB in culture skin cells
Compared to untreated MC, UVB (25 mJ/cm2) significantly
increased BMP6, ALK3, ALK6 and BMPRII expression in fully-
matched monocultures of MC and KC at the gene (using semi-
quantitative and qPCR) and protein levels (by immunofluorescence;
Fig. 2A–C). By contrast, UVB irradiation resulted in inhibition
of the anti-melanogenic BMP4 at gene and protein level in these
cells. However, UVB had opposite effects on the expression of
the BMP antagonists, whereby it stimulated BMP4 antagonist
noggin (NOG) but inhibited BMP6 antagonist sclerostin (SOST;
Fig. 2A–C). Further confirmation that these MC were UVB-
responsive was provided by the expected upregulation of MITF
and tyrosinase (TYR) and melanin transfer-associated markers
CDC42 and MYOX (Scott et al., 2002; Singh et al., 2010)
(Fig. 2A,B) after irradiation.
Fig. 1. Normal adult human MC express
BMP6 and BMP4 and their respective
antagonist Sclerostin and Noggin in situ.
Double immunolabelling of normal human
epidermis with anti-gp100 (NKI/beteb) (red) and
(A) anti-BMP6 antibody (green), (B) anti-
Sclerostin antibody (green), (C) anti-BMP4
antibody (green), (D) anti-Noggin antibody
(green), (E) anti-ALK3 antibody (green),
(F) anti-ALK6 antibody (green). Boxed areas are
shown at higher magnification on the right. The
top panels show MC and KC colocalisation as a
merged image (yellow, arrowheads) and the
middle and bottom panels individual
immunoprobes (middle: green BMP6, Sclerostin,
BMP4, Noggin, ALK3 and ALK6 expression in
MC and KC; lower panel: red, gp100 expression
in MC). Scale bars: 20 mm.
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BMP6 stimulates melanin synthesis by upregulatingtyrosinase expression and activity, and so works in anopposite manner to BMP4
Here we show the novel finding that BMP6 (at 1027 M) markedly
stimulated melanin production in MC, which was completely
inhibited by the BMP6 antagonist sclerostin (Fig. 3A). This effect
of BMP6 is likely related to its parallel and marked stimulation of
tyrosinase expression and to the dopa-oxidase activity of tyrosinase,
both of which were completely inhibited by sclerostin
(Fig. 3Bi,ii,Ci,ii). By contrast as previously reported by Yaar and
colleagues, BMP4 inhibited these pigment cell parameters (Yaar
et al., 2006), however we extend those findings to report that this
pigmentary inhibition could be lifted by co-stimulation of the MC
with the BMP4 antagonist noggin (Fig. 3).
BMP6 stimulates melanin transfer from MC to KC via a
MYOX-associated stimulation of filopodia formation in MC
In this study we show for the first time that melanin transfer
can be markedly upregulated by BMP6 in fully-matched
MC–KC co-cultures, when compared with unstimulated controls
(Fig. 4Ai,ii). Interestingly, this BMP6-induced upregulation of
melanin transfer was associated with a corresponding stimulation
of MC filopodia as observed by SEM analysis (Fig. 4Bi,ii). As
MYOX is known to mediate BMP6-induced filopodia formation in
other cells types (Pi et al., 2007), we attempted to better understand
this process by investigating its effect on MYOX expression in
MC. Exposure of adult human epidermal MC to BMP6 increased
filopodia formation by up-regulating MYOX (gene and protein) in
a dose- and time-dependent manner (Fig. 4C,D). Loss of MYOX
in MC, via the selective knockdown of the MYOX gene (confirmed
by RT-PCR, Fig. 5A), removed filopodia from MC (Fig. 5Bi,ii)
regardless of additional stimulation with the pro-melanogenic
BMP6 (Fig. 5Bi,ii) or the anti-melanogenic BMP4 (Fig. 5Bi,ii).
This finding indicates that MYOX is a key downstream regulator
of BMP-mediated filopodia formation in MC and supports the
findings that this may be a general strategy used also in other cell
types (Pi et al., 2007). To confirm the role of this atypical myosin
in BMP6-induced melanin transfer, we used MYOX knockdown
Fig. 2. UVB up-regulates the
expression of BMP6 and its receptors
in both MC and KC. (A) Normal human
MC exposed to 25 mJ/cm2 UVB and
incubated for 6 h in vitro; mRNA
expression of BMP6 and BMP receptors
ALK3, ALK6 and BMPR-II were
upregulated while expression of BMP6
antagonist sclerostin was reduced. BMP4
expression was reduced following UVB
irradiation, while expression of BMP4
antagonist noggin was increased. Parallel
positive controls for filopodia-associated
genes MYOX, CDC42, and for
melanogenic genes MITF, tyrosinase
were upregulated by UVB. qPCR data
are shown as fold change of expression
(basal conditions taken as 1). Values are
means 6 s.e.m. of three independent
experiments with ***P,0.0001,
**P,0.001, *P,0.01. (B) Semi-
quantitative RT-PCR for the detection of
BMP (BMP6, BMP4), BMP receptors
(ALK6, ALK3, BMPR-II), BMP
antagonists (SOST, NOG) filopodial
genes (MYOX, CDC42) and melanogenic
genes (MITF, TYR) in normal human
epidermal MC and KC with or without
exposure to 25 mJ/cm2 UVB for 6 h.
(C) MC or KC monocultures with or
without exposure to 25 mJ/cm2 UVB for
12 h. MC were double immunolabelled
with anti-tyrosinase antibody (red) and
anti-BMP6, anti-BMP4, anti-sclerostin,
anti-noggin and anti-Alk6 antibodies (all
green) to reveal the change in protein
expression in response to UVB. Scale
bars: 20 mm. KC monocultures were
similarly treated and double-
immunolabelled with anti-actin (red) and
anti-BMP6, anti-BMP4, anti-sclerostin,
anti-noggin and anti-Alk6 antibodies (all
green). Scale bars: 20 mm.
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MC (MYOX kd) to establish co-cultures with normal KC. BMP6-
induced melanosome transfer to KC was almost completely
blocked in these co-cultures as compared to control co-cultures
with intact MC MYOX (Fig. 5Ci,ii). We extend here the current
understanding of how BMP4 inhibits skin pigmentation by
showing that in addition to a tyrosinase-inhibiting effect, BMP4
also inhibited both MC filopodia formation and melanin transfer
from MC to KC (Fig. 5Ci,ii). These data provide important new
information that highlights the opposing effects of BMP4 and
BMP6 in these processes (Fig. 5Bi,ii,Ci,ii).
BMP6 stimulates melanin transfer from MC to KC via the
BMP6 receptors ALK3/6
The knockdown of ALK3/6 receptors in MC (ALK3kd6kd MC)
resulted in a significant loss of MC filopodia (as observed by SEM
analysis) compared with control MC (Fig. 5Bi,ii). This novel and
unexpected finding suggests that functional BMP receptors are
required for normal filopodia formation in MC, and that this is
likely due to MYOX effects on BMP receptor expression (Pi et al.,
2007). In this way, ALK3/6 receptor expression in MC appear to
be involved in at least some signalling pathway influencing
melanin transfer. To confirm this we used ALK3kd6kd MC to
establish co-cultures with normal KC, and show that melanosome
transfer was reduced in these co-cultures compared with controls
(Fig. 5Ci,ii). Neither BMP6 nor BMP4 influenced filopodia
formation or melanin transfer in the absence of ALK3/6
receptors on MC (Fig. 5Bi,ii,Ci,ii).
There is evidence that MYOX expression is not only
upregulated by BMP6 stimulation of MC (Fig. 4), but that
MYOX expression is actually required for maximal BMP signal
generation (Pi et al., 2007). To assess whether this interaction
between BMP receptors and MYOX expression also exists in adult
human epidermal MC, we examined the expression of MYOX in
MC lacking ALK3/6 and similarly also examined the expression of
ALK3/6 in MC lacking MYOX. The absence of MYOX resulted in a
marked reduction in ALK3/6 expression, and vice versa, compared
with control cells (Fig. 5A), revealing an important interaction
between MYOX and BMP receptors. Further stimulation of MC
with BMP6 did not significantly alter this interaction, suggesting
that the latter also operates under unstimulated basal conditions.
Fig. 3. BMP6 increases melanin synthesis in normal human epidermal MC by up-regulating tyrosinase expression and activity, while BMP4 shows the
opposite effect. Normal human epidermal MC in the presence or absence of BMP-4 (100 ng/ml) were treated with or without its selective antagonist, Noggin (400
ng/ml) for 72 h. MC were also treated in the presence or absence of BMP6 (100 ng/ml) with or without its selective antagonist, Sclerostin (400 ng/ml) for 72 h.
MC cells were treated with IBMX (100 mM) as a positive control. (A) Melanin content was determined spectrophotometrically (475 nm) after NaOH
solubilisation. Noggin fully blocked BMP4-associated reduction in melanin content, whereas Sclerostin completely inhibited the BMP6-induced melanin increase.
Results were expressed as percentage change in melanin content compared to unstimulated control. Values are means 6 s.e.m. of three independent experiments;
**P,0.001, ***P,0.0001. (B) (i) Protein extracts were electroblotted and membranes stained with L-DOPA for the estimation of tyrosinase activity. Noggin
fully blocked the BMP4-mediated decrease in tyrosinase activity, whereas sclerostin completely inhibited BMP6-induced tyrosinase activity. (ii) Densitometric
scanning of band intensities. Results are expressed as percentage increase compared to unstimulated controls. Values are means 6 s.e.m. of three independent
experiments; *P,0.05, **P,0.001. (C) (i) Western blot demonstrating changes in tyrosinase expression. BMP4-associated reduction in tyrosinase expression was
fully restored by co-incubation with noggin, whereas sclerostin completely inhibited BMP6-induced tyrosinase expression. (ii) Densitometric scanning of band
intensities from Ci. Results are expressed as fold increase compared to unstimulated control levels. Values are means 6 s.e.m. of three independent experiments;
*P,0.05, **P,0.001.
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BMP antagonists differentially regulate BMP-mediated
effects on melanin transfer in MC-KC co-culture
As the effect of BMP antagonists on filopodia formation and
melanin transfer have not been studied so far (neither for BMP4
nor BMP6), we were keen to determine whether BMP antagonists
also regulate BMP-mediated effects on MC filopodia formation
and melanin transfer to KC. The incubation of MC with BMP6 in
the presence of its antagonist sclerostin resulted in a marked
inhibition of BMP6-induced MC filopodia formation and melanin
transfer to KC (Fig. 6Ai,ii,Bi,ii, Fig. 4A,B). Similarly, noggin
significantly reversed the inhibitory effect of BMP4 on filopodia
formation and melanin transfer in MC (Fig. 6Ai,ii,Bi,ii).
BMP6 and BMP4 regulate the expression of filopodia-
associated proteins in MC
We show, using qPCR, that BMP6 stimulated the expression of
CDC42 and MYOX along with other filopodial markers such as
Fascin-1 (FSCN1), Vasodilator-stimulated phosphoprotein (VASP),
Integrinb1 (ITGB1) and Integrinb3 (ITGB3; Fig. 7A). By contrast
BMP4, inhibited the expression of these filopodial markers
(Fig. 7B). Both BMP6 and BMP4 stimulated the expression of
BMP receptors in MC, although induction of receptor expression was
more prominent with BMP6 (Fig. 7A,B). We also examined BMP
effects on the classical melanogenic genes MITF and TYR and found
that BMP6 increased expression of both genes (Fig. 7A), while
BMP4 significantly inhibited TYR but not MITF gene expression
compared to untreated control (Fig. 7B). We were interested to
examine the nature of the signalling pathway(s) involved in both the
melanogenic and melanin transfer/filopodial effects of BMPs in MC.
The SMAD pathway is involved in BMP6-induced melanin
transfer, but not in BMP6-induced melanogenesis
Phosphorylated (i.e. activated) receptor-regulated SMAD1/5/8
was detected with a nuclear and cytoplasmic distribution in both
KC and MC in intact human skin (Fig. 8Ai). By contrast,
inhibitory SMAD6 was detected mostly in the cell cytoplasm
(Fig. 8Aii). Strong SMAD6 expression was also detected in
cultured MC and KC, and even in their filopodia (Fig. 8Bi,ii).
To assess the involvement of the inhibitory Smad, SMAD6, in
both basal and BMP6-stimulated melanogenesis and melanin
transfer, we used siRNA to knockdown of the I-Smad, SMAD6, in
MC (Fig. 8Ci,ii). In the absence of SMAD6 (i.e. in SMAD6kd MC)
the phosphorylation of SMAD1/5/8 was increased (Fig. 8Ciiib)
compared to control MC (Fig. 8Ciiia). This finding suggests that
Fig. 4. Melanin transfer between MC and KC
is stimulated by BMP6 via the MYOX-
associated stimulation of filopodia formation.
(A) (i) Matched co-cultures of MC and KC
treated with increasing concentrations of BMP6
for 24 h. Double-immunolabelling with anti-
gp100 antibody (green) and anti-cytokeratin
antibody (red) showed a marked dose-dependent
increase in number of fluorescent spots
transferred to KC. Nuclei were counterstained
with DAPI. Scale bars: 20 mm (ii) Quantification
of melanosomes transferred to KC in Ai. Data
are means 6 s.e.m.; 20 cells/condition were
assessed in each of three independent
experiments. **P,0.001, ***P,0.0001.
(B) (i) SEMs of MC treated with 100 ng/ml
BMP6. Scale bars: 20 mm. High-power views
show BMP6-induced filopodia formation in MC
compared to untreated controls (lower panels).
(ii) Quantification of MC filopodia from the
SEMs in Bi. Data are represented as means 6
s.e.m.; 10 cells/condition were assessed in each
of three independent experiments; ***P,0.001.
(C) RT-PCR of MYOX (upper panel) and
western blot analysis (lower panel) of MYOX
expression in MC after BMP6 treatment at
increasing concentrations for 6 h. (D) RT-PCR
of MYOX (upper panel) analysis and western blot
(lower panel) of MYOX expression in MC after
BMP6 (100 ng/ml) treatment at indicated time-
points. GAPDH was used as a loading control for
RT-PCR and Actin as a loading control for
western blots.
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SMAD6 is constitutively active in MC under basal conditions,
and so could interfere with the activation of BMP receptors. BMP6
treatment of control MC resulted in the up-regulation of SMAD6
gene expression (Fig. 8Cii), and so this I-Smad may interfere
with the activation of receptor SMAD1/5/8. SMAD1/5/8
phosphorylation was higher in BMP6-treated MC compared with
untreated control MC (Fig. 8Ciiic,iiia), and was also higher in
BMP6-treated SMAD6-knockdown MC compared to BMP6-
treated control MC (Fig. 8Ciiid,iiic).
To investigate the involvement of the Smad pathway in BMP6-
induced melanogenesis, we assessed the level of receptor
SMAD1/5/8 phosphorylation in SMAD6kd versus SMAD6
control MC in the presence/absence of BMP6 (Fig. 8Ciii,D).
We speculated that if the Smad pathway contributed to BMP6-
induced melanogenesis, then the loss of SMAD6 (i.e. associated
with greater SMAD1/5/8 activation; see Fig. 8Ciii) would result
in a further increase in BMP6-induced melanogenesis. However,
we found no significant difference in the level of BMP6-induced
melanogenesis in the absence of SMAD6 (Fig. 8D). Similarly,
we extended the work of Park et al. to show that there was also no
significant difference in the melanogenesis-inhibitory effects of
BMP4 in the absence of SMAD6 (Fig. 8D) compared to cells
treated with control siRNA (Park et al., 2009b).
Next, we asked whether Smad signaling plays a role in melanin
transfer from MC to KC. Here knockdown of inhibitory SMAD6 in
MC was used to assess whether SMAD6 loss resulted in enhanced
BMP6-induced melanin transfer from MC to KC. To do this we
established a co-culture with Smad6kdMC partnered with normal
KC. These co-cultures exhibited enhanced melanosome transfer to
KC compared with co-cultures established with Smad6ctrl MC and
Fig. 5. The selective knockdown of MYOX and ALK3/6 receptor expression in MC inhibits BMP6-induced melanin transfer from MC to KC. (A) Semi-
quantitative RT-PCR for endogenous MYOX, ALK3 and ALK6 was performed in presence or absence of BMP6 (100 ng/ml) in MC treated with synthetic siRNAs
targeting human MYOX, ALK3 and ALK6 or non-silencing control siRNA. DNA size markers are in the left lane, and GAPDH was used as a loading control.
(B) (i) The dorsal surface of MC treated with (a–c) control siRNA: (a) basal condition – numerous filopodia are present; (b) BMP6 treated – filopodia are induced;
(c) BMP4 treated – filopodia are reduced compared to untreated control. (d–f) The dorsal surface of MC treated with MYOX siRNA exhibited an almost complete
inhibition of filopodia formation irrespective of BMP treatment. (g–i) The dorsal surface of MC treated with ALK3/6 siRNA exhibited a reduction of filopodia
after treatment with either BMP6 or BMP4, compared with control siRNA-treated MC grown under basal conditions. Scale bars: 5 mm. (ii) Filopodia per cell were
counted from SEM images of normal MC treated with control siRNA, MYOX siRNA or ALK3/6 siRNA either in the presence or absence of BMP6 or BMP4.
Values are means 6 s.e.m.; 10 cells/condition were assessed in each of three independent experiments. *P,0.01, **P,0.001, ***P,0.0001. (C) Normal MC
treated with siRNAs targeting MYOX, ALK3/ALK6 and non-silencing control siRNA. These MC were used to establish fully matched co-cultures with normal KC.
(i) Double-immunolabelling of co-cultures for gp100 (NKI/beteb; green) and cytokeratin (red) revealed clear changes in number of transferred green fluorescent
spots (i.e. melanin granules transferred to KC). MC–KC co-cultures established with control siRNA MC: (a) numerous gp100-positive spots are seen in KC in the
basal condition; (b) increased numbers of gp100-positive spots are transferred after BMP6 treatment; (c) a reduced number of gp100-positive spots are transferred
after BMP4 treatment. (d–f) MC–KC co-culture established with MYOX siRNA-treated MC exhibited almost complete inhibition of gp100-positive spots
transferred to KC irrespective of BMP treatment. (g-i) MC–KC co-culture established with ALK3/6 siRNA-treated MC exhibited a marked reduction in gp100-
positive spots transferred to KC in both BMP6- and BMP4-treated MC compared with control siRNA-treated MC–KC co-culture. Scale bars: 20 mm.
(ii) Quantification of melanosomes transferred to KC. Data are represented as means 6 s.e.m.; 20 cells/condition were assessed in each of three independent
experiments. *P,0.01, **P,0.001, ***P,0.0001.
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KC (Fig. 8Ei,ii); this level of transfer was further enhanced in the
presence of BMP6 (Fig. 8Eii). These findings suggests that
Smad1/5/8 activation plays a crucial role in melanin transfer to
KC under both basal and BMP6-stimulated conditions.
p38 MAPK is involved in both BMP6-induced
melanogenesis and BMP6-induced melanin transfer in
human skin cells
As we have shown above that BMP6-induced melanogenesis
appears to be independent of the Smad pathway, we next
investigated the potential involvement of the MAPK and PI3-K
pathways in both melanogenesis and melanin transfer using
MAPK and PI3-K inhibitors. Inhibition of p38MAPK (by
SB203580) markedly diminished BMP6-induced melanogenesis
to below basal levels (Fig. 9A), whereas MAPKK (MEK)
inhibition (by PD98059) and PI3-K inhibition (by LY294002)
enhanced melanogenesis (Fig. 9A). SB203580 also decreased
melanogenesis in MC not treated with BMP6, confirming the
previously reported role of p38 MAPK in melanogenesis (Singh
et al., 2005). Furthermore, the observed enhancement of basal
or BMP6-induced melanogenesis by PD98059 or LY294002
(Fig. 9A) indicates that the ERK1/2 and PI3-K pathways can
negatively regulate melanogenesis as we have shown previously
(Singh et al., 2005).
BMP6 stimulated p38MAPK phosphorylation and its
translocation to the cell nucleus in both MC and KC (Fig. 9B),
and this was inhibited by SB203580 (Fig. 9B). Consistent with
this, we also found that BMP6 induced MITF and TYR mRNA
expression in MC (Fig. 9C), which was blocked by the
p38MAPK inhibitor SB203580 (Fig. 9C). Given that BMP6-
associated effects on melanin transfer were dependent on Smad
pathway activation (Fig. 8), we next determined whether this
additionally involves activation of the MAPK or PI3-K pathways.
The incubation of matched MC–KC co-cultures with either
SB203580 and LY294002 markedly reduced melanosome
transfer from MC to KC (Fig. 9Di,ii). However, inhibition of
the ERK1/2 pathway (by PD98059) failed to significantly inhibit
melanin transfer under both basal and BMP-6 stimulated
condition (Fig. 9Di,ii), suggesting that melanin transfer is
regulated independently ERK1/2 activation.
DiscussionA role for BMP6 in human skin pigmentation had not been
evaluated so far. Here we find that normal adult human skin
expresses BMP6 and BMP4, and their respective receptors and
antagonists, indicating that adult skin is very likely a BMP6-
responsive tissue. Moreover, BMP6 stimulated melanin synthesis
in normal; adult epidermal melanocytes by upregulating tyrosinase
Fig. 6. The BMP antagonists sclerostin and noggin inhibit
BMP6- and BMP4-mediated melanin transfer effects.
(A) (i) (a) In the basal condition the dorsal surface of MC
exhibited numerous filopodia. (b) MC treated with BMP-6 (100
ng/ml) and sclerostin (400 ng/ml) for 24 h exhibited reduced
filopodia formation. (c) BMP4-treated (24 h) MC exhibited
reduced filopodia formation. (d) MC treated with BMP-4 (100 ng/
ml) and Noggin (400 ng/ml) for 24 h exhibited increased
filopodia formation compared to untreated control MC. High-
power views of the boxed regions are shown in the right panels.
Scale bars: 20 mm. (ii) Filopodia/cell were counted from SEM
images of normal MC treated with control siRNA, MYOX siRNA
and ALK3/6 siRNA either in the presence or absence of BMP6 or
BMP4. Values are means 6 s.e.m.; 10 cells/condition were
assessed in each of three independent experiments; **P,0.001.
(B) (i) Double-immunolabelling of matched MC–KC co-culture
for gp100 (NKI/beteb; green) and cytokeratin (red) revealed clear
changes in the numbers of green fluorescent spots transferred to
KC after different stimulations (24 h). (a) In the basal condition
numerous gp100-positive spots were transferred to KC. (b) BMP4
(100 ng/ml)-treated cells exhibited a reduction of gp100-positive
spots transferred to KC. (c) Sclerostin (400 ng/ml)-treated cells
exhibited a reduction of gp100-positive spots transferred to KC.
(d) Noggin (400 ng/ml)-treated cells exhibited an induction of
gp100-positive spots transferred to KC. (e) Cells treated with
BMP6 (100 ng/ml) and sclerostin (400 ng/ml) exhibited a
reduction of gp100-positive spots transferred to KC. (f) Cells
treated with BMP4 (100 ng/ml) and noggin (400 ng/ml) exhibited
increased gp100-positive spots transferred to KC compared with
the untreated MC–KC co-culture. Scale bars: 20 mm.
(ii) Quantification of melanosomes transferred to KC. Data are
represented as means 6 s.e.m.; 20 cells/condition were assessed
in each of three independent experiments. *P,0.01,
**P,0.001, ***P,0.001.
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expression and activity, and worked in an opposite manner to
BMP4 as reported earlier by Yaar et al. (Yaar et al., 2006). This
effect is likely related to its parallel and marked stimulation of
tyrosinase expression and of the dopa-oxidase activity of
tyrosinase, both of which were completely inhibited by the
BMP6 antagonist sclerostin. Thus, we identify BMP6 as a potent
melanogenic stimulator in epidermal MC.
As previously reported UVB irradiation increased melanin
synthesis in normal human adult epidermal MC (Friedmann and
Gilchrest, 1987). These changes are thought to occur via UVB
effects on pro/anti-melanogenic auto-/paracrine factors. Here
we found that the expression of BMP6 and its receptors is
upregulated by UVB in both MC and keratinocytes. Thus, this
system in skin cells is highly responsive to UVB irradiation and
in this way BMP6 may behave similarly to other potent
melanogens, including a-melanocyte-stimulating hormone (a-
MSH), which contribute to UV-induced tanning. By contrast,
UVB irradiation inhibited expression of anti-melanogenic BMP4
in both MC and KC but had opposite effects on BMP4 antagonist
(i.e. noggin) expression. These opposing effects of BMPs indicate
an unforeseen complexity to the regulation of MC by different
members of the BMP family.
UVR also stimulates MC dendricity, filopodia formation andmelanin transfer (Scott et al., 2002; Singh et al., 2010). However,
a significant gap remains in our understanding of just howmelanin transfer is controlled at steady state conditions in skin.Physiological factors such as pro-opiomelanocortin-derivedpeptides (e.g. a-MSH) (Virador et al., 2002) have been
implicated. Here we for the first time showed that melanintransfer and filopodia formation can be markedly stimulated byBMP6 incubation in fully-matched MC–KC co-cultures.
Filopodia have been implicated in organelle transport (e.g.vesicles) (Rustom et al., 2004; Vidulescu et al., 2004), and theatypical myosin, MYOX, is known to regulate filopodia
formation (Bohil et al., 2006; Tokuo et al., 2007). MYOX is akey downstream regulator of BMP-induced filopodia formationin MC and melanosome transfer to KC, as confirmed by thealmost complete blocking of melanin transfer in MC with MYOX
knockdown. By contrast, BMP4 inhibited both MC filopodiaformation and melanin transfer from MC to KC. These dataconfirm a prominent role for MYOX in BMP6-induced MC
filopodia formation and melanin transfer to KC, and highlightsagain the opposing effects of BMP4 and BMP6 in theseprocesses.
We for the first time also show that BMP6 stimulates melanintransfer from MC to KC via the BMP6 receptors ALK3/6, asknockdown of ALK3/6 resulted in a significant loss of MC
filopodia. This finding suggests that functional BMP receptorsare required for formation of normal filopodia in MC, and thatthis is likely due to MYOX effects on BMP receptor expression(Pi et al., 2007). Interestingly, there was a marked reduction in
ALK3/6 expression in the absence of MYOX, and vice versa,revealing an important interaction between MYOX and BMPreceptors. These data indicate that MYOX is not only required for
filopodia formation, but is also required for BMP6 receptor-mediated MC activation, by amplifying subsequent BMPresponses. This provides an explanation for how MYOX may
participate in BMP6-induced melanin transfer to KC.
BMP effects on cellular targets depend on the availability oftheir extracellular antagonists (Balemans and Van Hul, 2002).Here we found that BMP antagonists differentially regulate
BMP-mediated effects on melanin transfer in MC–KC co-culture.The incubation of MC with BMP6 in the presence of itsantagonist sclerostin resulted in a marked inhibition of BMP6-
induced MC filopodia formation and melanin transfer to KC,while noggin significantly reversed the inhibitory effect of BMP4on filopodia formation and melanin transfer in MC. These data
not only suggest a novel role for sclerostin as a melanogenesisand melanin-transfer inhibitor, but also demonstrate that nogginmay act as a stimulator of melanogenesis and melanin transfer in
human epidermis. In this way, our data complements andexpands a previous report showing that noggin competes forbinding to BMP receptors and can darken coat colour in mice(Sharov et al., 2005).
MYOX is thought to act downstream of the GTPase familymember CDC42, a master regulator of filopodia formation andmelanin transfer (Scott et al., 2002; Singh et al., 2010). Here we
show that BMP6 upregulates the expression of several filopodia-associated proteins FSCN1, VASP, ITGB1 and ITGB3 in MC,while BMP4, inhibited this. Our findings that several genes
involved in filopodia formation were also differentially regulatedby BMP6 and BMP4 can be interpreted in the context of MYOX-associated cell substrate adhesion (via integrins) and actin
Fig. 7. BMP6 and BMP4 regulate BMP receptor and filopodial-
associated gene expression. (A) BMP6 upregulates mRNA expression of
MYOX, FASCIN-1, DDC42, VASP, INTB1, INTB3 and BMP receptors ALK3,
ALK6 and BMPR-II in MC at 6 h. These qPCR data are shown as fold
expression (basal condition taken as 1). Values are means 6 s.e.m. of three
independent experiments; *P,0.001, **P,0.0001. (B) BMP4 downregulates
mRNA expression of BMP proteins MYOX, FASCIN-1, CDC42, VASP,
INTB1, INTB3 but upregulates those of BMP receptors ALK3, ALK6 and
BMPR-II in MC at 6 h. These qPCR data are shown as fold expression (basal
condition taken as 1). Values are means 6 s.e.m. of three independent
experiments; *P,0.001, **P,0.001, ***P,0.0001; NS, not significant.
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Fig. 8. The Smad pathway is involved in BMP6-induced melanin transfer, but not in BMP6-induced melanogenesis in normal human skin cells.
(A) Double-immunolabelling analysis of normal human epidermis with anti-gp100 (NKI/beteb; red) and anti-phosphoSMAD 1/5/8 antibody (green; i). Boxed area
is also shown as a higher-power view in the upper panel (merged image), which shows in situ nuclear localisation of phosphorylated SMAD1/5/8 (green) in KC
(arrowheads), and in gp100-positive MC (red) of human epidermal skin and the individual immunoprobes (lower: green phospho-SMAD 1/5/8). (ii) Anti-SMAD6
antibody (green). The boxed area is also shown as a high-power view in the upper panel (merged image), which shows colocalisation (yellow, arrowheads), and
individual immunoprobes (lower: green SMAD6). Inhibitory SMAD6 was mostly detected in KC and MC cytoplasm in human epidermis. Scale bars: 20 mm.
(B) Double-immunolabelling of (i) MC with anti-Smad6 antibody (green) and anti-tyrosinase (red), and (ii) KC with anti-SMAD6 antibody (green) and actin (red).
Strong expression of Smad6 was detected in both MC and KC. Boxed areas are shown as higher power views (right panel: Smad6 expression in filopodia). Scale
bars: 20 mm. (C) (i) Normal MC treated with siRNAs targeting SMAD6 and non-silencing control siRNA. Double-immunolabelling with anti-SMAD6 antibody
(green) and anti-tyrosinase antibody (red) revealed a complete inhibition of SMAD6 expression in MC. (ii) Semi-quantitative reverse transcription-PCR for
endogenous SMAD6 mRNA was performed in the presence or absence of BMP6 (100 ng/ml) on normal human MC treated with synthetic siRNAs targeting human
SAMD6 and non-silencing control siRNA. DNA size markers are in the left panel; GAPDH was used as a loading control. (iii) Normal MC treated with siRNAs
targeting SMAD6 and non-silencing control siRNA. Double-immunolabelling with anti-phosphoSMAD1/5/8 antibody (green) and anti-tyrosinase antibody (red)
revealed a nuclear localisation/activation of phosphoSMAD1/5/8. Scale bars: 20 mm. (D) Normal MC treated with siRNAs targeting SMAD6 and non-silencing
control siRNA. Cells were incubated with or without BMP6 (100 ng/ml) or BMP4 (100 ng/ml) for 72 h. Melanin content was determined spectrophotometrically
(475 nm) after NaOH solubilisation. Results were expressed as the percentage change in melanin content compared to unstimulated control. Values are means 6
s.e.m. of three independent experiments; NS, not significant. (E) (i) Normal MCs treated with siRNAs targeting SMAD6 and non-silencing control siRNA. These
MC were used to established co-culture with normal KC. Double-immunolabelling of SMAD6 (red; a,c) and gp100 (red/green b,d) revealed a complete inhibition
of SMAD6 expression in MC, leading to a clear stimulation in the number of green fluorescent spots transferred to KCs (white arrow). (ii) MC–KC co-cultures as
shown in Ei were incubated with or without BMP6 (100 ng/ml), and melanosomes transferred to KC were quantified. Data are represented as means 6 s.e.m.; 20
cells/condition were assessed in each of three independent experiments. *P,0.01, **P,0.001.
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filament elongation due to the anti-capping activity of Ena/VASP
(Trichet et al., 2008; Menna et al., 2009). These results provide a
mechanistic rationale for the opposing roles of distinct BMPs in
MC filopodia formation and melanin transfer to KC.
Despite the overall high levels of homology between different
BMPs, significant differences in their bioactivities have been
demonstrated in different tissues. For example, in chronic kidney
disease BMP2 appears to induce osteoblastic differentiation of
vascular smooth muscle cells leading to vascular calcification,
while BMP7 demonstrates opposing effects (Hruska et al., 2005).
These opposing effects can be explained by BMP receptor
preferences and the potential utilization of type I heterodimers.
BMP2/4 ligands require a heterodimer of type I receptors,
whereas BMP5/6/7 ligands signal exclusively through type I
homodimers (Lavery et al., 2008). Other potential determinants
of whether BMP activities parallel or diverge from each other in
any given tissue might be the tissue-specific pattern of BMP
receptor expression.
We were interested to examine the nature of the signalling
pathway(s) involved in both the melanogenic and melanin
transfer/filopodial effects of BMPs in MC. BMP signalling is
modulated by numerous proteins at various points. Once the
BMP signal is transduced into the intracellular compartment it
can be modulated by the activation of inhibitory Smad proteins
(I-Smads). I-Smads, including SMAD6 and SMAD7, function as
antagonists of another group of Smads called receptor-regulated
Smads (R-Smads, e.g. SMAD1/5/8). I-Smads interact with BMP
type I receptors that have been activated by type II receptors
through their MH2 domains, though these I-Smads are not
released from type I receptors and thus prevent the activation of
the R-Smads (Miyazono, 2008). Smad6 preferentially represses
BMP signalling by inhibiting signals from BMP type I receptors,
Fig. 9. p38 MAPK is involved in both BMP6-induced melanogenesis in MC and melanin transfer from MC to KC. (A) The effect of specific kinase
inhibitors on BMP6-induced melanogenesis in MC (72 h) was assessed. MC were incubated with or without BMP6 (100 ng/ml) in the presence or absence of
SB203580 (SB; 10 mM), PD98059 (PD; 10 mM) and LY294002 (LY; 10 mM). Melanin content was determined spectrophotometrically (475 nm) after NaOH
solubilisation. Results were expressed as the percentage change in melanin content compared to unstimulated control. Data are means 6 s.e.m. of three
independent experiments; **P,0.001, ***P,0.0001. (B) MC and KC monocultures were treated with or without BMP6 (100 ng/ml) in the presence or absence of
SB203580 (SB; 10 mM) (2 h). Cells were double-immunolabelled with anti-phospho-p38 MAPK (green) or anti-p38 MAPK (green) and anti-tyrosinase antibody
(red) to reveal nuclear translocation/activation of p38 MAPK. Scale bars: 20 mm. (C) Semi-quantitative RT-PCR for time-course expression of MITF and TYR
mRNA in MC in response to BMP6 (100 ng/ml) in the presence or absence of SB203580. GAPDH was used as a loading control. (D) (i) The effect of specific
kinase inhibitors on BMP6-induced melanin transfer in matched MC–KC co-culture was assessed. Cells were incubated with or without BMP6 (100 ng/ml) for 24
h in the presence or absence of SB203580 (SB; 10 mM), PD98059 (PD; 10 mM) and LY294002 (LY; 10 mM). Cells were double-immunolabelled with anti-gp100
antibody (NKI/beteb; green) and anti-cytokeratin (red), and revealed clear changes in number of green fluorescent spots transferred to KC. Scale bars: 20 mm.
(ii) Quantification of melanosomes, shown in Di, transferred to KC. Data are means 6 s.e.m.; 20 cells/condition were assessed in each of three independent
experiments. *P,0.001, **P,0.001, ***P,0.0001. NS, not significant.
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while Smad7 inhibits both TGF-b and BMP signalling (Goto
et al., 2007). We show here that the SMAD pathway is involved
in BMP6-induced melanin transfer but not in melanogenesis.
These results suggest that the Smad pathway may not play any
significant role in BMP-mediated regulation of melanin
synthesis. It is also unlikely that a Smad-dependent pathway is
directly involved in the upregulation of MITF-M transcripts in
MC, because the promoter region of MITF gene does not contain
a DNA consensus sequence for Smad binding sites (Shibahara
et al., 2001). By contrast, we report here that Smad1/5/8
activation plays a crucial role in melanin transfer to KC under
both basal and BMP6 stimulated conditions.
BMPs have been shown to activate ERK1/2, p38MAPK and
PI3-K in numerous cell types (Miyazono et al., 2010; Osyczka and
Leboy, 2005; Nohe et al., 2002; Javelaud and Mauviel, 2005).
Here we found p38MAPK to be involved in BMP6-induced
melanogenesis in human skin cells. We also found that BMP6
induced MITF and TYR mRNA expression in MC, was blocked by
the p38MAPK inhibitor, validating our view that p38MAPK plays
an important role in BMP6-induced melanogenesis, and concurs
with previous reports that BMPs stimulate MITF and TYR
protein expression in chick embryo retinal pigment epithelium
(Muller et al., 2007). Stress signalling, mediated via p38MAPK
phosphorylation, is known to result in a rapid and persistentphosphorylation of Ser307 of MITF, which we and others have
shown to be responsible for the transcription of genes involved inMC differentiation, proliferation and survival (Mansky et al.,2002; Vance and Goding, 2004; Saha et al., 2006). Furthermore,UVR-induced activation of stress-responsive p38MAPK can also
lead to the phosphorylation of the ubiquitous bHLH-LZtranscription factor, USF-1, and like MITF this can bind andactivate the TYR promoter in human MC (Galibert et al., 2001). It
is likely therefore, that BMP6 and UVR-mediated phosphorylationof p38MAPK results in the phosphorylation of target transcriptionfactors such as MITF and USF-1, which are able to bind and
activate the TYR promoter.
Whether the observed BMP6-associated effects on melanintransfer were dependent on Smad pathway activation and whetherthis additionally involves the activation of the MAPK or PI3-K
pathways is of interest. SB203580 and LY294002 markedlyreduced BMP6-induced melanosome transfer from MC to KC,suggesting that p38MAPK and PI3-K are involved in BMP6-
mediated melanin transfer. Moreover, inhibition of the ERK1/2pathway failed to significantly inhibit melanin transfer, suggestingthat melanin transfer is regulated independently ERK1/2
activation. We have not evaluated the role of p38MAPK andPI3-K in the filopodia formation in human MC; however, thesepathways were found to be involved in the regulation of filopodia
formation in other cell lines (Gadea et al., 2004; Vadlamudi et al.,1999; Adam et al., 1998). The involvement of PI3-K in BMP6-induced melanin transfer substantiates our recently proposed viewof how MC filopodia interact with KC phagocytosis during the
melanin transfer process (Singh et al., 2010). Here the motorprotein MYOX, a recognised effector of phagocytosis, acts as amolecular link between PI3-K activation and pseudopodia
extension during phagocytosis (Cox et al., 2002).
When taken overall data from this study suggest that BMP6 up-regulates melanogenesis and melanin transfer in normal adult
human skin cells, in marked contrast to other BMPs (e.g. BMP4and BMP2) (Fig. 10) (Yaar et al., 2006). These importantpigmentary functions can be additionally modulated by theBMP-selective antagonists sclerostin and noggin. Thus, the net
level of melanin transfer from MC to KC in human skin, whichunderpins the UVR-protective nature of skin pigmentation, isinfluenced via a delicate balance of BMPs and their selective
antagonists. BMP6-mediated effects on pigmentation arecontrolled by complex signalling events. Prominent among theseare the stress-responsive p38MAPK pathway that can regulate
melanogenesis in human MC independently of the Smad pathway,and a combination of p38MAPK, PI3-K and Smad pathways whichare involved in melanin transfer between MC and KC. This
supports an increasing appreciation that facultative melanogenesisis likely a ‘stress response’ to UVR (Galibert et al., 2001; Plonkaet al., 2009), where the photoprotective response of facultativemelanogenesis or tanning is functionally similar to the SOS
response described in other species including bacteria (Eller andGilchrest, 2000). Here we present evidence that DNA damagestimulates pigmentation, at least in part, through up-regulation of
tyrosinase mRNA and protein levels.
Both major MC functions (i.e. melanogenesis and melanintransfer) have the potential to be differentially regulated via non-
overlapping signalling pathways, i.e. Smad pathway involvementin melanin transfer but not melanogenesis. It may well ofinteresting to also investigate whether the BMP system is
Fig. 10. A proposed model for melanin transfer and melanogenesis in
human melanocytes involving the ‘canonical’ BMP–SMAD pathway and
the BMP–MAPK pathway. In the former, BMP6 dimers bind to the receptor
complex, leading to phosphorylation of the type I receptor (RI) by the type II
receptor (RII), which in turn phosphorylates R-SMAD (SMAD1, -5, -8). This
phosphorylation enables R-SMAD to complex with co-SMAD (SMAD4).
Then the SMAD4 and R-SMAD/SMAD4 complex enters the nucleus to
activate target genes such as those for the filopodial proteins, MYOX,
CDC42, FASIN, VASP and ITGB3 to induce melanin transfer. SMAD6
inhibits the BMP–SMAD pathway. SMAD6 is induced by BMP signalling,
establishing negative feedback loops. Alternatively, in the BMP–MAPK
pathway, activated BMP receptors can activate p38 MAPK, which in turn
activates target genes including MITF. Activated MITF can induce the
transcription of tyrosinase to stimulate melanogenesis. Activated BMP
receptors can also activate PI3-K and p38 MAPK to stimulate BMP6-
mediated melanin transfer. BMP6 effects on cellular targets depend on the
availability of its extracellular antagonists such as sclerostin.
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involved in the biogenesis, maturation and distribution of otherlysosome-related organelles, in the context of general cellularhomeostasis.
Materials and MethodsMaterials
Recombinant human BMP6, BMP4, Noggin, Sclerostin were from R&D Systems(Minneapolis, MN, USA). Inhibitor SB203580 was from Sigma, while InhibitorsPD98059 and LY294002 were from Cell Signaling Technology, Inc. (Beverly,MA, USA). Antibodies to Smad6, MyoX, BMP6 , BMP4 antibody, Sclerostin andNoggin were from Abcam, (Cambridge, UK), while phospho-Smad 1/5/8 fromMillipore (Billerica, MA, USA) and tyrosinase were from Santa CruzBiotechnology, (Santa Cruz, CA, USA).
Matched epidermal melanocyte/keratinocyte co-culture
Human abdominal skin was obtained with informed consent and local researchethics approval from normal healthy Caucasian donors with skin photo-type II(n55, female 39–67y, average 51y) after elective plastic surgery. All cell culturereagents were from Invitrogen Ltd. (Paisley, UK) unless stated otherwise.Epidermal melanocytes (MC) cultures were established as previously described(Singh et al., 2010; Kauser et al., 2003) and grown in keratinocyte (KC) serum-freemedium (K-SFM) with Eagle’s minimal essential medium (EMEM) supplementedwith 1% FBS, 16 non-essential amino acids, penicillin (100 U/ml)/streptomycin(100 mg/ml), 2 mM L-glutamine, 5 ng/ml basic fibroblast growth factor, and 5 ng/ml endothelin-1 (Sigma, Dorset, UK).
Matched epidermal KC were established from the same biopsy specimen as theMC above (Singh et al., 2008) and grown in K-SFM supplemented with 25 mg/mlbovine pituitary extract (BPE), 0.2 ng/ml rEGF, penicillin (100 U/ml)/streptomycin(100 mg/ml), and 2 mM L-glutamine. Culture medium was replenished every secondday. KC and MC were identified using anti-cytokeratin antibody (Abcam,Cambridge, UK) and melanocyte-specific NKI/beteb antibody (Monosan, Uden,The Netherlands) to gp100, respectively. For co-culture studies MC (passage 3) andKC (passage 2) were seeded onto Lab-TekH chamber slides (ICN Biomedicals Inc.,Aurora, OH, USA) at 46104 cells/well and in 1 MC to 10 KC ratio (Singh et al.,2008). Analysis of melanosome organelle transfer was performed at 24 h. Forexperiments, MC or MC–KC co-culture were treated BMP6 or BMP4 with orwithout their antagonists sclerostin or noggin. For some experiments, MC or KC orMC–KC co-culture were treated with inhibitors SB203580 (SB; 10 mM), PD98059(PD; 10 mM) and LY294002 (LY; 10 mM) in the presence or absence of BMP6.
UV irradiation
MC or KC monocultures were irradiated with UVB as previously described (Singhet al., 2010). Briefly, cells were cultured in ‘starved’ medium lacking FBS and BPE(i.e. retaining bFGF and endothelin-1 for MC viability), temporarily submerged inPBS and irradiated with 25 mJ/cm2 UVB using a fluorescent UVB lamp (WaldmannUV6; emission 290–400 nm, peak 313 nm; Herbert Waldmann GmbH, Villingen-Schwenningen, Germany). UVR consisted of 66% UVB and 34% UVA. The PBSwas removed immediately after irradiation and replaced with fresh ‘starved’ media.Control cells were treated similarly but not irradiated. MC were analysed byquantitative PCR (qPCR) and semi-quantitative RT-PCR after 6 h UVB irradiationto evaluate BMP, BMP receptor and BMP antagonist gene expression. The proteinswere assessed by double immunolabelling in MC and KC monoculture after 25 mJ/cm2 UVB exposure.
SEM assessment of cell morphology
MC monoculture was prepared for SEM as described previously (Singh et al.,2010). Briefly, cells were fixed with 1% glutaraldehyde at 37 C, post-fixed in 1%osmium tetroxide and 1% tannic acid as mordant, dehydrated through a series ofalcohol (20% to 70%), stained in 0.5% uranyl acetate, followed by dehydration(90% and 100%) before final dehydration in hexamethyl-disilazane (Sigma,Dorset, UK) and air-drying. Each slide was gold sputter-coated (EMITECH, K550)(Blazer 20 mA) for 10 min. Specimens were viewed under field emission SEM(FEI Quanta 400, Eindhoven, The Netherlands) at 10 keV.
Immunofluorescence confocal microscopy
Double-immunofluorescence staining in MC or KC monocultures, MC–KC co-culture, and human skin cryosections was performed as described previously(Singh et al., 2010). Briefly, cells and tissue were fixed in ice-cold methanol for 10min before air drying and rehydration in PBS before blocking with 10% donkeyserum (90 min) before overnight incubation at 4 C with primary antibodies NKI/beteb (1:30), BMP6 (1:50), BMP4 (1:50), Sclerostin (1:50), Noggin (1:200), Alk3(1:50), Alk6 (1:50), phosphoSmad1/5/8 (1:50), Smad6, phosphop38MAPK andp38MAPK followed by incubation with Alexa488-conjugated secondary antibody(1:100; Invitrogen, Paisley, UK) for 1 h. The second primary antibodies totyrosinase (1:100; Santa Cruz Biotechnology, CA, USA) or cytokeratin (1:100;Abcam, Cambridge, UK) were applied for 1 h followed by a Alexa594-conjugated
secondary antibody (1:100; Invitrogen, Paisley, UK). Slides were mounted in 49,6-diamidino-2-phenylindole (DAPI)-containing medium (Vector, Peterborough, UK)and imaged on a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany).
Melanin assay
Melanin levels were assessed as described previously (Kauser et al., 2003). Briefly,human MC (26105) were treated with either vehicle controls, or stimulants for 72h, washed, trypsinised and counted before pelleting. Melanin/cell was quantifiedafter boiling in 1 M sodium hydroxide (NaOH) and read against synthetic melanin(Sigma, UK) at 495 nm.
DOPA oxidase activity of tyrosinase
MC (26105) were incubated with test compounds, vehicle control or IBMX(161024 M) for 72 h, prepared for SDS-PAGE (un-reduced and un-boiled proteinextract) and transblotted onto PVDF membranes. The latter were incubated at RTwith 5 mM L-DOPA in 0.1 M sodium phosphate buffer for 3 h, before stopping thereaction in distilled water before scanning PVDF membranes.
siRNA knockdown of MYOX, ALK3/ALK6, SMAD6 in MC
MC monocultures or fully-matched MC–KC co-cultures were transfected withsiRNA according to the manufacturer’s instructions (Invitrogen, Paisley, UK).Briefly, 1 day prior to siRNA treatment the cells were incubated in 37 C, 5% CO2
for 12 h to allow cell attachment. The following synthetic siRNAs (Qiagen, WestSussex, UK) were used: Felxitube Gene solution for MYOX, Entrez gene ID:4651(4 siRNAs) (cat. no. GS4651; NM_012334, length of transcript: 11436 bp);BMPR1B, Entrez gene ID:658 (4 siRNAs) (cat. no. GS658; NM_01203, length oftranscript: 5560 bp); BMPR1A, Entrez gene ID:657 (4 siRNAs) (cat. no. GS657;NM_004329, length of transcript: 3631 bp); SMAD6, Entrez gene ID:4091 (4siRNAs) (cat. no. GS4091; NM_001142861, length of transcript: 1293 bp). MYOXor BMPR1A/1B or SMAD6 siRNA (25 nM) or control siRNA (25 nM) (non-homologous to mammalian genome) was incubated with Lipofectamine 2000(Invitrogen, Paisley, UK) for 20 min to allow complex formation, before additionto co-cultures. Transfection medium was replaced after 12 h with complete mediaand at 24 h post-siRNA transfection ‘knockdown’ was verified by doubleimmunofluorescence using antibodies against TYR and SMAD6. Parallel sampleswere assayed by RT-PCR to verify knockdown.
For some experiments MYOX siRNA-treated MC or BMPR1A/1B siRNA-treatedMC were processed by SEM in order to test the siRNA effects on filopodia. TheseMC were used to establish co-culture with normal KC to study their effects onmelanosome transfer. For co-culture studies control siRNA and MYOX siRNA-treated MC or BMPR1A/1B siRNA-treated MC or SMAD6 siRNA-treated MC (at12 h) were seeded with untreated normal KC in chamber slides at 46104 cells/wellin a ratio of 10 normal KC to 1 siRNA-treated MC. These co-cultures wereprocessed at 36 h by double labelling with gp100 (NKI/beteb) and cytokeratinantibody to detect melanosome transfer to KC.
Semi-quantitative RT-PCR analysis
RT-PCR was performed as previously described (Singh et al., 2010). Briefly,RNA was extracted from MC cultures using RNeasy isolation kit (Qiagen, WestSussex, UK) according to the manufacturer’s instructions and quantified in aspectrophotometer at 260 nm. cDNA synthesis was performed with 2 mg of totalRNA using Superscript III First Strand Synthesis Super Mix (Invitrogen, Paisley,UK). The primer sequences, PCR products and their annealing temperature aresummarized in supplementary material Table S1. Cycling conditions were used at95 C for 15 min; 96 C for 15 s; 60 C 15 s and 72 C for 30 s for 30 cycles. Afteramplification, 10 ml of the reaction mixture was loaded onto a 1% agarose gel(Sigma, Poole, UK) and electrophoresed (Sigma, Poole, UK). A 100–1000 basepair DNA ladder (Invitrogen, Paisley, UK) was also loaded.
Quantitative PCR amplification
qPCR amplification commenced with 1 ml purified cDNA being added to 24 mlreaction mixture; 12.5 ml QuantiTect SYBR Green RT-PCR Kit (Qiagen, WestSussex, UK), 9 ml RNase free water and 2.5 ml of each primer (supplementarymaterial Table S2) (Qiagen, West Sussex, UK). Thermal cycling conditions were95 C for 15 min; 96 C for 45 s, 58 C for 45 s and 72 C for 30 s, for 45 cycles. Thehouse-keeping gene, recombinant 18S, was used as a control for RNA loading ofsamples and the MyiQ 2.0 Optical System Software was used for analysis ofSYBR Green I stained PCR products (Bio-Rad Laboratories Ltd, Hertfordshire,UK).
Western blotting
Western blot analysis was done as previously described (Singh et al., 2010).Briefly, protein (35 mg per well) was separated by 8% SDS-PAGE under reducingconditions and electroblotted onto PVDF membranes (Immobilon, Millipore,Bedford, MA, USA), and immunoprobed overnight at 4 C with antibodies againstTYR (1:200), MYO-X (1:200) and ACTIN (1:1000; Santa Cruz Biotechnology,CA, USA), followed by incubation for 2 h at RT with a horseradish-peroxidase-
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conjugated donkey anti-sheep/goat IgG antibody (1:600; Serotec Ltd, Kidlington,
Oxford, UK) or a horseradish peroxidase-conjugated donkey anti-rabbit IgGantibody (1:1000; GE Healthcare, Chalfont St Giles, Buckinghamshire, UK). The
reactions were detected by the Enhanced Chemiluminescence plus kit (AmershamBiosciences Ltd, Buckinghamshire, UK).
Quantitative analysis of melanosome transfer
This was performed as previously described (Singh et al., 2008). Briefly, MC–KCco-culture slides were fixed in ice-cold methanol for 10 min at 220 C, washed in
PBS and then blocked with 10% donkey serum. First primary antibody NKI/beteb(1:30) was applied overnight at 4 C, followed by incubation with Alexa-Fluor-488-
conjugated secondary antibody (Invitrogen, Paisley, UK) (1:100) for 1 h at room
temperature. The second primary antibody against cytokeratin (1:100) was appliedfor 1 h at room temperature followed by a Alexa-Fluor-594-conjugated secondary
antibody (1:100) (Invitrogen, Paisley, UK). Slides were mounted in DAPI-
containing medium (Vector, Peterborough, UK) and imaged on a Zeiss LSM 510confocal microscope (Carl Zeiss, Jena, Germany). Evaluation of melanosome
transfer MC–KC co-cultures were performed by counting fluorescent gp100-positive spots within recipient KC in 5 random microscopic fields per well at 606magnification in each of three independent experiments (i.e. a total minimum of 60
KC). This generated an average spot value per KC for each of three independentexperiments, which were themselves averaged and final values presented as 6
s.e.m. Different cells will show different numbers of internalize melanosomes,
which can be due to a range of factors like proximity to MC as well as cell volumeetc. To avoid counting melanin granules that may still be associated with MC, we
only counted gp100-positive spots within KC that were not in direct contact withMC.
Measurements of filopodia
This was performed as previously described (Singh et al., 2010). Slender
cylindrical structures on the dorsal surface of cells were counted as filopodia if
they had a diameter of <0.1 mm and .1 mm in length (Pi et al., 2007). Filopodiawere counted by viewing the images at high power to discriminate between closely
positioned and overlapping filopodia (especially those which had partially
collapsed on the cells apical surface) on 10 individual cells per treatment andthe average number of filopodia/cell was calculated 6 s.e.m.
Statistical analysis
Statistical analysis was performed using Student’s paired t-test. Quantitative data
are presented as means 6 s.e.m. for three separate experiments. Statistically
significant differences are denoted with asterisks: *P,0.01, **P,0.001 and***P,0.0001.
FundingThis research received no specific grant from any funding agency inthe public, commercial or not-for-profit sectors.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.102038/-/DC1
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