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First published online 5 December 2007
doi: 10.1242/dev.011031
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Cutaneous Biology Research Center, Harvard Medical School and Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA.
* Author for correspondence (e-mail: bruce.morgan{at}cbrc2.mgh.harvard.edu)
Accepted 15 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Corin, Dermal papilla, Agouti, Pigmentation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the active growth phase, the mature hair follicle is composed
primarily of keratinocytes arranged in concentric layers of differentiated
cell types that comprise the hair shaft (HS), inner root sheath (IRS) and
outer root sheath (ORS). Growth of the hair occurs as proliferating cells in
the hair matrix at the base of the follicle generate additional constituents
of the inner layers of the follicle that are organized into the IRS and HS and
extruded through the ORS towards the surface of the skin. The DP plays a
central role in this process. The keratinocytes abutting the DP act as stem
cells of the hair bulb, undergoing asymmetrical divisions to generate transit
amplifying (TA) progeny, which then undergo a few divisions before terminally
differentiating (Legue and Nicolas,
2005
). Ablation and grafting studies have demonstrated that the DP
is required to maintain the growth of the hair follicle and suggest that it
plays an instructive role in driving morphogenesis of the hair
(Jahoda et al., 2001;
Oliver and Jahoda, 1988).
This period of sustained hair growth lasts for a few weeks in the mouse. At
the end of this growth or anagen phase, proliferation in the hair bulb ceases.
During the degeneration or catagen phase, the matrix cells either terminally
differentiate or apoptose, and the basal end of the hair shaft becomes
anchored in the upper follicle. The outer root sheath from the lower
two-thirds of the follicle degenerates and the DP is drawn to the base of the
permanent portion of the follicle. A quiescent or telogen phase ensues that
may last from a few days during the first hair cycle to many weeks in mature
animals. At the end of the telogen phase, keratinocyte stem cells resident in
the bulge region of the permanent follicle are activated and regenerate the
lower portions of the anagen follicle
(Morris et al., 2004). It is
thought that the DP plays a central role in the activation of these
keratinocytes and the subsequent guidance of their proliferation and
differentiation to regenerate the follicle, although the nature of that role
remains to be empirically defined (Sun et
al., 1991).
In addition to its functions in follicle morphogenesis and cycling, the DP
also regulates pigmentation of the hair. Pigment is synthesized by melanocytes
resident in the hair bulb and deposited in the keratinocytes of the hair
medulla and cortex. Pigment production in melanocytes is regulated in part by
the activity of the Mc1r receptor expressed on their surface. When this
receptor is active, the black pigment eumelanin is produced and deposited in
the hair shaft. Mc1r has a basal level of constitutive activity in the absence
of its agonists melanocortins that is further stimulated by the binding of
these ligands (Barsh, 1999;
Smart and Low, 2003). Although
the DP is likely to play a role in generating an environment that attracts and
maintains melanocytes in the hair bulb
(Yoshida et al., 1996), it
also regulates hair pigmentation more directly by expressing the agouti
signaling protein, hereafter referred to as agouti
(Millar et al., 1995). Binding
of agouti to Mc1r reduces its activity and results in a shift from the
production of eumelanin to the synthesis of pheomelanin, a yellow pigment
(Barsh et al., 2000;
Chai et al., 2003).
Agouti (also known as nonagouti or ASIP) is expressed
transiently in the DP of the dorsal pelage during the early growth phase of
the hair cycle. The resultant provisional switch to pheomelanin deposition
generates a subapical yellow band in the otherwise black hair that defines the
agouti coat color. Despite the predominance of black pigment in the hair of
agouti mice, the presence of lighter pigment in the hair tip creates the
overall appearance of a mottled brown hair coat that provides adaptive
coloration in the natural environment. Modest variations in the length of this
apical pheomelanin band can dramatically alter coat appearance and represent
one mechanism by which adaptive changes in coat color can occur
(Hoekstra, 2006).
Partly as a result of the development of mouse strains that allow the
manipulation of gene expression in the embryonic precursors of the epidermis
and follicular epithelium (Byrne et al.,
1994; Indra et al.,
1999), the epidermal placode
(Levy et al., 2005) or the
stem cells of the adult hair follicle (Ito
et al., 2005), significant progress has been made in understanding
the genetic mechanisms that direct follicle formation in its keratinocyte
constituents. Despite its functions in maintaining the niche for matrix stem
cells and in organizing both the morphogenesis and pigmentation of the hair
follicle, the molecular genetics of DP formation and function have remained
less accessible to study because of a comparative lack of tools to purify
these cells or manipulate gene expression in vivo. To address this need, we
evaluated genes preferentially expressed in the DP of the hair follicle to
identify those whose expression in the skin is restricted to this population.
Here we report the identification of Corin as a gene specifically
expressed in the DP of the hair follicle. Ablation of Corin activity
in the DP reveals that it is not required for hair follicle morphogenesis and
confirms that the Corin gene is well suited to serve as a platform
for the manipulation of gene expression in the DP in vivo. This analysis also
reveals an unexpected function for this transmembrane serine protease in the
regulation of hair shaft pigmentation and identifies a novel mechanism by
which the DP directs the pattern of pigmentation in the hair shaft.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In-situ hybridization and immunohistochemistry
Non-radioactive in situ hybridization of sections from embryos and dorsal
skins were performed with probes corresponding to nts 682-1252 of
Corin (GenBank Acc. No. NM_016869) and nts 126-613 of Agouti
(NM_015770). Anti-Corin antibodies (1:800) raised in rabbits
(Fig. 2A), were detected with
FITC-conjugated secondary antibody (1:250) (Jackson) in the presence of
TO-PRO-3 (1:40,000).
Hair shaft analysis
The analysis of the length of the subapical yellow band was performed on
six wild-type and seven mutant postnatal day (P)20 mice. At P20, hairs derived
from the first hair cycle are fully grown and are the only hairs present in
the dorsal pelage. For each mouse, approximately 400 hairs from mid-dorsal
pelage were randomly mounted on slides in a thin layer of Gelvatol. Awl hairs
represent approximately 10% of the total hair population. Therefore, about 80
awl hairs per mouse were specifically collected for analysis. Two-tailed
unpaired t-tests were performed.
Hair shafts were photographed in bright field and in green fluorescent channel at 100 x magnification. Lack of black pigment in the hair shaft results in autofluorescence that corresponds with the deposition of yellow pigment. The green channel of the fluorescent image was duplicated in the red channel to generate a yellow color. The modified fluorescent image was overlaid on the bright field image in PhotoShop and reduced to 45% opacity.
Real-time PCR
Mid-dorsal skins from P0-P9 mice were collected and used to prepare RNA
with Trizol solution (Invitrogen). The total RNA was further purified using
RNeasy mini kit (Qiagen) and a DNase I digestion step. Normalized RNA was
reverse transcribed using random hexamer primers. Primer pairs (Superarray)
for Actb (PPM02945A), Corin (PPM41062A), Agouti
(PPM24722A), Mc1r (PPM04903A), Pomc (PPM37114A),
Atrn (PPM30947A) and Mgrn1 (PPM02945A) and CYBR
Green-Fluorescein PCR Mater Mix were used with an iCycler (Bio-Rad), MyiQ
Single-color Detection system, MyiQ Optical System Software. Differences
between samples were quantified based on the 
Ct method. The
number of mice per genotype (WT/Mut) per stage used in this expression profile
was as follows: P0(5/3); P1(9/10); P2(13/10); P3(8/7); P4(13/9); P5(7/11);
P6(5/6); P7(3/4); P8(2/2); P9(4/5). The analysis was performed individually
for each mouse and the average values were calculated for each group of mice.
Thus, the s.d. reflects the variation in gene expression between individual
mice in the same group.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A
population enriched in DP cells was purified from newborn skin on the basis of
GFP expression (Shimizu and Morgan,
2004). Gene expression in these cells immediately after isolation
was compared with that of cells maintained in culture with the expectation
that genes expressed in the DP as a consequence of inductive signaling from
the follicular keratinocytes would be preferentially lost from the cultured
cell expression profile. Among the genes identified in this way was the
Corin locus. Corin transcripts were readily detected in
freshly isolated cells but dropped below the level of detection within 24
hours in culture (see Fig. S1 in the supplementary material). Corin
encodes a transmembrane protease that is expressed in the heart and
participates in blood pressure regulation by cleaving the prohormone proANP to
its active form (Chan et al.,
2005; Yan et al.,
2000). EST expression profiles and available expression data
suggested that Corin expression was largely restricted to
cardiomyocytes (see also Yan et al.,
1999). This restricted expression profile and the expression of
the Corin protein on the surface of the cell made this gene an attractive
candidate for further investigation of its expression and function in the
skin. Although mice lacking Corin have the altered blood pressure regulation
and cardiovascular defects predicted by its role in cleaving ANP, no skin or
hair phenotypes were reported in these mice
(Chan et al., 2005).
Corin expression in the hair follicle
Hair follicle formation occurs in three waves during embryogenesis, which
initiate at E14, E16 and between E18 to birth. These waves give rise to
follicles that generate distinct hair types. Follicles that give rise to the
long, straight guard hairs arise in the first wave, whereas the
intermediate-sized awl and smaller zigzag and auchene hair types are thought
to arise in the second and third waves, respectively. Corin
expression at the RNA level was examined in embryonic skin and in the dorsal
pelage of post-natal animals by in situ hybridization
(Fig. 1). It is first detected
in the skin in the nascent dermal condensate at embryonic day (E)15, as the
first wave of follicles form (Fig.
1A). Its expression persists in the DP throughout the development
of these follicles and is also observed in the dermal condensate and DP of the
second and third waves of follicles throughout their morphogenesis
(Fig. 1B,C). Corin transcripts
were not detected elsewhere in the skin at any stage examined. Corin
expression persists and remains restricted to the DP throughout the anagen
(growth) phase of the hair cycle (Fig.
1D-G and data not shown). Expression is not detected during the
catagen (regression) or telogen (resting) phases but returns with the onset of
a new anagen phase (data not shown). Corin is expressed in a similar
pattern in all follicle types of the dorsal pelage.
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Corin is not required for hair growth or cycling
A null allele of Corin was generated by inserting a YFP-Neo
cassette into the first exon of Corin that harbors the initiation
codon and encodes most of the intracellular N-terminal domain
(Fig. 3A). Mice homozygous for
this allele (Corin-/-) were recovered at expected
frequencies and were viable and fertile
(Fig. 3B-D). Immunostaining
with anti-Corin antibodies confirmed both the absence of detectable Corin
protein in these mice and the specificity of the anti-sera used in this study
(Fig. 3E,F). In P3 skin, the
three waves of follicle formation are represented by follicles in different
stages of development. All three stages of hair follicles are present in
mutant skin with frequencies similar to that of the wild type, suggesting that
hair follicle initiation is normal in homozygous mutants
(Fig. 3E,F, see Fig. S2 in the
supplementary material). Indeed, the hair coat in homozygous mutant animals
appears normal with respect to numbers, frequency, structure and growth rate
at all stages examined (Fig. 4
and see Fig. S3 in the supplementary material). Corin activity is not required
for the normal morphogenesis or cycling of the hair follicle.
Corin modifies agouti activity
However, there is a striking difference between the wild-type and mutant
pelage. Homozygous mice exhibit a distinctly lighter coat color that is most
pronounced in juveniles but persists through adulthood
(Fig. 4A,B). This phenotype is
dependent on the presence of a functional allele (A) of agouti.
Corin mutants homozygous for a null allele of the agouti gene
(a/a) are black and indistinguishable from the wild type
(Fig. 4C), whereas
Corin mutants on A/a or A/A backgrounds are
progressively lighter than corresponding mice with a wild-type Corin
allele (Fig. 4D). The yellow
appearance is a result of pheomelanin production and not alteration of the
diffractive properties of the hair, because tryosinase mutants incapable of
synthesizing melanin have white fur irrespective of the presence or absence of
Corin (Fig. 4C). Heterozygotes
for Corin are indistinguishable from the wild type on all
agouti backgrounds tested (data not shown).
To gain more insight on the mechanism underlying the coat color phenotype, hair shafts were plucked from the back skin of wild-type and mutant mice at the end of the first hair cycle. The four types of hair, guards, awls, zigzags and auchenes, differ in the extent of pheomelanin content. Changes in hair type could in principle explain the lighter coat color. However, all four types of hair were present at similar frequencies in both wild-type and mutant mice (see Fig. S3 in the supplementary material). Corin is not required to specify hair type.
The length of the subapical yellow band was analyzed separately for each
hair type. Zigzag hairs were the most abundant hair type and have a prominent
yellow band. Regardless of Corin genotype, all zigzag hairs exhibited
a yellow band that was restricted to the apical segment of the hair. In mutant
zigzag hairs, the length of the yellow band was increased.
Fig. 5A shows extreme examples
of wild-type and mutant zigzag hairs: in the absence of Corin the yellow band
extended for the length of the terminal segment, whereas in the wild type it
extended less than half of that length. Both the length of the terminal
segment and the length of the yellow band vary between different zigzag hairs
on the same mouse. To quantify the differences between wild-type and mutant
zigzag hairs, the ratio (R) between the length of the yellow band (Y) and the
length of the apical segment (Z) was calculated
(Fig. 5B). Hairs were scored
for the presence or absence of a black tip and assigned to one of three
categories: R
0.5, 0.5<R0.75, or 0.75<R1
(Fig. 5C-H). This analysis
revealed two major differences (see Fig.
5I). First, the basal extension of the yellow band increased
substantially in the mutant. The number of zigzag hairs with R>0.5
increased significantly from 33% in the wild type to 85% in mice lacking
Corin, and the category exhibiting R>0.75 increases from 0 to 32%. Second,
the yellow band was also extended in the apical direction in most hairs.
Although virtually all of the wild-type hairs had a terminal black tip, 70% of
the mutant hairs did not (see Fig. S4 in the supplementary material). It is
striking that the extension of the yellow band in the apical and basal
directions are to some extent independent events. Hairs with the longest
yellow bands may nevertheless have a black tip, whereas hairs in the shortest
category may have yellow tips. Despite the breadth of the categories used in
this analysis, the pigmentation pattern of a minimum of 85% of the zigzag
hairs is altered in the Corin mutant.
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0.25 and
0.25<R0.5. Representative examples of all three categories are shown in
Fig. 6A. A striking reduction
in the frequency of completely black awl hairs from 61% in the wild type to
28% in the mutant was observed (Fig.
6C). In the absence of Corin, many awl hairs that otherwise would
be completely black were transformed to include a subapical yellow band.
Furthermore, the percentage of awl hairs with 0.25<R0.5 increased from
7% in the wild type to 54% in mutant mice. This increase (47%) exceeds the
proportion that could be contributed by awl hairs that normally exhibit a
yellow band in wild type (39%) and demonstrates that hairs that would normally
lack any discernible agouti activity show pheomelanin deposition over a
quarter of their length in the absence of Corin. A similar elongation of the yellow band was observed in auchene hairs as a result of Corin ablation (see Fig. S5 in the supplementary material). As observed in zigzag hairs, the extension of the pheomelanin band in the apical and basal directions were independent events. Although substantial extension in the basal direction was observed, all mutant auchene hairs end with black tips as observed in wild-type mice. In contrast to zigzags, awls and auchenes, all guard hairs were completely black in both wild-type and mutant mice, suggesting that agouti signaling is completely inactive in this type of hair, regardless of the presence or absence of Corin (data not shown).
The expression of agouti pathway genes is not altered in Corin mutants
The extent of the yellow band is regulated in part by the limited window in
which the agouti gene is expressed in dorsal skin
(Millar et al., 1995;
Vrieling et al., 1994), and
Corin could in principle modulate signals that impinge on DP cells to regulate
the expression of agouti. Detailed analysis of agouti expression by
real-time PCR during the hair cycle in wild-type mice was performed to define
the pattern of agouti expression
(Fig. 7B). Agouti
transcript levels were extremely low at P0 but rose rapidly from P1 to P3 and
then dropped off dramatically to baseline levels by P7. No changes in the
levels or timing of agouti transcript accumulation were detected in
Corin mutant mice. This whole skin analysis preferentially detected
the expression of agouti in zigzag hairs that comprise 70-80% of the dorsal
pelage. Therefore, agouti expression was also evaluated by in situ
hybridization to score expression in guard and awl hair follicles
(Fig. 7G). As predicted by the
lack of a pheomelanin band in Corin mutant guard hairs, agouti
expression was not detected in guard hair follicles in either wild-type or
mutant mice at any of the time points evaluated, which included P0-P6
(Fig. 7G and see Fig. S6 in the
supplementary material). However, agouti transcripts were readily
detected in the DP of some, but not all awl hair follicles at P2 and later
stages. No change in the pattern, timing or apparent levels of agouti
transcript accumulation was observed in Corin-/- skin, and
expression in both second and third wave follicles of wild-type and mutant
mice was largely extinguished between P6 and P7.
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-melanocyte stimulating hormone (-MSH) to Mc1r
competitively inhibits agouti binding
(Ollmann et al., 1998). By
either mechanism, a decrease in -MSH levels would be expected to
enhance pheomelanin production on an Agouti background. Although
Pomc (the gene encoding the precursor of -MSH) is mainly
expressed in the brain, it is also expressed at low levels in the skin and in
cultured keratinocytes (Slominski and
Paus, 1993). Therefore, Corin may act on the environment
surrounding the DP to control mRNA levels of Pomc. However, similar
low levels of Pomc expression were detected in the skin of wild-type
and Corin mutant mice throughout anagen
(Fig. 7D). In addition to Mc1r,
the activity of agouti is dependent on at least two other proteins expressed
by the melanocyte, attractin (Atrn) and mahogunin (Mgrn1)
(Gunn et al., 1999;
He et al., 2003a;
He et al., 2003b;
Phan et al., 2002). Atrn is a
single-pass transmembrane protein that binds agouti, and Mgrn1 is an E3
ubiquitin ligase. Although the mechanism by which these proteins modify
pigment production remains unknown, the prevailing model is that the
simultaneous binding of agouti to Atrn and Mc1r activates Mgrn1 to catalyze
ubiquitylation events targeting Mc1r for modifications that decrease its
activity, possibly by promoting its degradation or internalization
(He et al., 2003a). In
contrast to agouti and Mc1r, which are predominantly
expressed in the DP and the melanocyte respectively, Atrn and
Mgrn1 are more widely expressed. The mRNA levels of Atrn and
Mgrn1 were stable throughout the anagen phase of the hair cycle and
remained unchanged regardless of Corin genotype
(Fig. 7E,F).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Corin ablation reveals cryptic agouti expression
These results also demonstrate that Corin plays an unexpected role
in modifying the balance between eumelanogenesis and pheomelanogenesis. This
activity is exerted in concert with the agouti pathway. In the absence of a
functional allele of agouti there is no discernible effect on pigment
production. It is noteworthy that Corin expression precedes agouti
signaling activity and persists long after the decline of detectable agouti
activity, whereas the phenotypic effects of Corin ablation are
observed at the start and/or the end of agouti signaling period. As
Corin expression at the RNA level coincides with protein detection of
Corin throughout the anagen phase of the hair cycle (compare Figs
1 and
2), translational regulation
does not explain the failure of Corin to suppress pheomelanogenesis
during the entire period of agouti expression. Therefore, Corin is
only able to effectively counteract agouti activity when the levels of agouti
are low, at the peripheries of its bell-shaped curve of expression. This
conclusion is supported by the observation that coat color is sensitive to the
dosage of agouti in the presence of Corin. The length of the
pheomelanin band is reduced when agouti dosage is halved, presumably
because a level of agouti sufficient to overcome Corin-mediated inhibition is
only reached closer to the peak period of agouti expression. The fact
that coat color is also sensitive to the dose of agouti in the
absence of Corin further demonstrates that agouti levels are limiting for
pheomelanin production at the edges of its period of expression.
The ablation of Corin does not alter the timing or level of agouti expression, or the expression of other known components of the agouti signaling pathway. Instead, ablation of Corin unmasks cryptic agouti activity. This cryptic activity is revealed in in situ analysis of agouti expression in wild-type skin, where hair follicles containing both agouti transcripts in the DP and melanocytes producing eumelanin in the adjacent hair bulb are readily detected. By contrast, the deposition of pheomelanin and the expression of agouti transcripts in the DP are well correlated in Corin-/- skin. In the mutant mice, all DP with detectable agouti expression are embedded in hair bulbs that do not exhibit eumelanin synthesis.
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Corin acts downstream of agouti mRNA expression
These results demonstrate that Corin acts downstream of agouti
gene transcript accumulation to functionally counteract agouti protein
activity. As a protease tethered to the surface of the DP cell, the Corin
protein is well positioned to regulate agouti protein activity in several
ways. One known Corin substrate is the prohormone Nppa, which when cleaved to
its active form, contributes to blood pressure regulation by activating the
natriuretic peptide pathway (Chan et al.,
2005; Wu et al.,
2002; Yan et al.,
1999; Yan et al.,
2000). Although we can detect the expression of the Nppb
and Nppc genes that encode related natriuretic peptide precursors in
the skin at low levels by PCR, neither these genes nor the signaling receptors
Npr1 and Npr2 are detectable in the hair follicle by in situ hybridization
under conditions that readily detect the robust expression of Nppa
and Nppb in the heart (data not shown). Although we cannot rule out
the Npp family as substrates that mediate this effect, alternative mechanisms
seem more likely.
Among these, a direct protein-protein interaction with a component of the
agouti pathway is a strong possibility. As secreted or transmembrane proteins,
agouti, -MSH (Pomc1), Atrn and Mc1r are all potential targets whose
activity could be attenuated (agouti or Atrn) or augmented (Mc1r or
-MSH) by the extracellular Corin protease activity. Of these,
-MSH is unlikely to be the target of Corin. Although Pomc1-null mice
have lighter coat color, particularly in the ventral skin, they do not
resemble the dramatic effect on coat color observed in Corin-deficient mice
(Challis et al., 2004;
Yaswen et al., 1999). By
contrast, the agouti protein is clearly limiting for the pheomelanin switch in
wild-type animals (Miller et al.,
1997). The fact that it is secreted from the DP, where Corin
levels are high, also makes agouti an attractive candidate for modification by
this protease.
The fact that the N-terminal and C-terminal domains of agouti bind
separately and independently to Atrn and Mc1r, respectively, and the failure
of these two domains to rescue in trans the activity of full-length agouti
(He et al., 2001;
Ollmann and Barsh, 1999),
suggest that binding of agouti to both Atrn and Mc1r forms a ternary complex
that is required for agouti signaling. If so, a proteolytic cleavage that
physically separates the N-terminal domain from the C-terminal domain would
not only render the agouti protein inactive but could also produce competitive
antagonists that interfere with agouti binding to both Mc1r and Atrn.
Agouti binding to Mc1r is conformation dependent. Cis and trans conformers
with markedly different abilities to compete for Mc1r binding are generated
when the C-terminal domain of agouti is synthesized in vitro
(McNulty et al., 2005). The
fact that agouti protein can assume different conformations with distinct
binding capabilities suggests that activity of agouti protein could also be
modified by a proteolytic cleavage that promotes a conformational change that
reduces or abolishes the affinity of agouti protein for either Mc1r, Atrn or
both.
In ventral skin, where agouti levels remain high and promote pheomelanin
production throughout the hair cycle, the majority of agouti protein detected
by western analysis migrates at the position expected of the full-length form
(Ollmann and Barsh, 1999).
Unfortunately, the low and variable level of agouti protein in dorsal skin,
the limited sensitivity of available reagents, and the partial nature of the
predicted cleavage events prevent direct assessment of these potential
cleavage models in dorsal skin in vivo.
Although mechanisms in which Corin acts directly on agouti might limit the
impact of these observations on human pigmentation, where a postulated role
for agouti in pigmentation based on linkage analysis remains
controversial, they might still have other implications for human health.
Ectopic expression of agouti in the brain induces obesity by acting
on the Mc4r that is normally inhibited by the agouti-related protein (Agrp)
(Duhl et al., 1994;
Manne et al., 1995). Aspects
of these two signaling pathways are distinct. First, agouti activity in the
brain is dependent on attractin and mahogunin
(He et al., 2001) whereas Agrp
activity is not (He et al.,
2003a; Ollmann et al.,
1997). Second, although the C- and N-terminal domains of agouti
must act in concert to inhibit Mc1r activity in vivo, binding of the
C-terminal domain of Agrp to its receptor is sufficient for function
(Ollmann et al., 1997).
Furthermore, the N-terminal domain of Agrp inhibits this function and must be
removed by proteolytic cleavage to activate Agrp
(Creemers et al., 2006;
Jackson et al., 2006). Thus,
cleavage between the N- and C-terminal domains is likely to have opposite
effects on Agouti and Agrp activity, but other possible proteolytic
modifications may have similar effects on both pathways. Although
Corin is not expressed in the CNS
(Yan et al., 1999), it is
possible that an analogous serine protease modulates the activity of Agrp
signaling and thus participates in the process of weight control and energy
balance. Whether Corin acts by modifying some component of agouti signaling or
by a natriuretic peptide pathway-related mechanism that antagonizes Mc1r
responses in melanocytes, these results identify a novel pathway to modulate
Mcr activity. Identifying the postulated analogous serine protease in the
brain would increase the repertoire of targets for drug development and allow
the exploration of new therapeutics against obesity.
Finally, variations in coat color have played a prominent role in
discussions of adaptation to the environment and the mechanisms that drive
evolution. Although a large number of genes contribute to pigmentation either
through functions in the generation, migration or maintenance of melanocytes,
biosynthesis of eumelanin, or transfer of pigment to recipient cells,
comparatively few have been identified that contribute to the
pheomelanin/eumelanin switch that plays such a significant role in coat
appearance
(http://www.cbc.umn.edu/ifpcs/micemut.htm).
We note that the Corin mutation reproduces the described differences
in agouti band length seen in deer mouse populations adapted to lighter
colored environments than their forest-dwelling counterparts
(Hoekstra, 2006). The
observations that levels of agouti signaling are limiting in the switch to
pheomelanin production and are effectively regulated by Corin only when agouti
activity is marginal identifies a novel route to the phenotypic changes
associated with adaptive coloration changes. Loss-of-function mutations in
Pomc cause a similar albeit less dramatic lightening of the dorsal
pelage, but also cause obesity and adrenal insufficiency
(Krude et al., 1998;
Yaswen et al., 1999). Loss of
Corin function may also incur other fitness costs, as both modest
hypertension and cardiac hypertrophy are observed in mutant mice, but these
costs are less dramatic (Chan et al.,
2005). Furthermore, if pigmentation and cardiovascular regulation
use different substrates, these two processes can be uncoupled by
substrate-specificity mutations (Knappe et
al., 2004). These attributes, and the lack of other genetic
modifiers downstream of agouti expression, suggest that an analysis of the
relative contribution of Corin mutations to adaptive changes in
pigmentation in wild populations will be informative.
In conclusion, these studies identify the transmembrane serine protease Corin as a novel modifier of the agouti signaling pathway that acts downstream of agouti gene expression to suppress agouti protein activity on the pheomelanin/eumelanin switch. The tight restriction of Corin expression to the DP during the anagen phase of the hair cycle within the skin, its relative lack of expression elsewhere and the apparent restriction of its activity to the regulation of pigment patterning, all suggest that this gene will serve as a useful platform to manipulate gene expression in the DP. This will broaden its contribution to the understanding of hair follicle biology beyond these important insights into the regulation of coat color.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/217/DC1
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ACKNOWLEDGMENTS |
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