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OCR for page 57
Colloquium
Inclian hecigehog and ~e-catenin signaling: Role in the
sebaceous lineage of normal and neoplastic
mammalian epidermis
C. Niemann*t, A. B. Unden*t, S. Lyle§, Ch. C. Zouboulis~, R. Toftgardt, and F. M. Watttll
Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; "Department of Biosciences at Novum, Karolinska Institutet, SE-141 57
Huddinge, Sweden; §Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and Department of Dermatology, University
Medical Center Benjamin Franklin, The Free University of Berlin, 14195 Berlin, Germany
In mammalian epidermis, the level of ,8-catenin signaling regu-
lates lineage selection by stem cell progeny. High levels of
,8-catenin stimulate formation of hair follicles, whereas low
levels favor differentiation into interfollicular epidermis and
sebocytes. In transgenic mouse epidermis, overexpression of
,`3-catenin leads to formation of hair follicle tumors, whereas
overexpression of N-terminally truncated Lef1, which blocks
,8-catenin signaling, results in spontaneous sebaceous tumors.
Accompanying overexpression of ,6-catenin is up-regulation of
Sonic hedgehog (SHH) and its receptor, Patched (PTCH/Ptch). In
ANLef1 tumors Ptch mRNA is up-regulated in the absence of
SHH. We now show that PTCH is up-regulated in both human and
mouse sebaceous tumors and is accompanied by overexpression
of Indian hedgehog (IHH). In normal sebaceous glands IHH is
expressed in differentiated sebocytes and the transcription
factor GLI1 is activated in sebocyte progenitors, suggesting a
paracrine signaling mechanism. PTCH1 and IHH are up-regulated
during human sebocyte differentiation in vitro and inhibition of
hedgehog signaling inhibits growth and stimulates differentia-
tion. Overexpression of ANLef1 up-regulates IHH and stimulates
proliferation of undifferentiated sebocytes. We present a model
of the interactions between ,6-catenin and hedgehog signaling
in the epidermis in which SHH promotes proliferation of pro-
genitors of the hair lineages whereas IHH stimulates prolifera-
tion of sebocyte precursors.
Mammalian skin is maintained by stem cells whose daugh-
ters differentiate along the lineages of the hair follicle,
interfollicular epidermis (IFE), and sebaceous gland (1-3~.
The stem cells are common targets for neoplastic conversion,
and the range of different types of epithelial tumor reflects
aberrant differentiation along the different epidermal lin-
eages. Thus, whereas basal cell carcinomas (BCCs) may reflect
the relatively undifferentiated phenotype of the hair follicle
outer root sheath, squamous cell carcinomas exhibit elements
of IFE differentiation, sebaceous tumors contain terminally
differentiated sebocytes, and pilomatricomas and trichofol-
liculomas contain cells undergoing differentiation along hair
lineages (44.
Many of the molecules that regulate epidermal self-renewal
and differentiation have now been identified (1, 2~. A key role
has emerged for I3-catenin signaling in response to the diverse
repertoire of Wnts expressed in different regions of the epider-
mis (5, 6~. When I3-catenin levels are elevated by expressing a
stabilized, N-terminally truncated form of the protein, there is de
novo formation of hair follicles in postnatal IFE (74. Conversely,
when I3-catenin is absent, or its activity blocked with dominant
negative forms of the downstream transcription factor Lefl, hair
follicles are converted into cysts of IFE with associated sebocytes
(8-10~. It thus appears that the level of 13-catenin controls
www. pnas.org/cg i/doi/ 10.1 073/pnas. 1834202100
lineage selection in the skin, with high levels promoting hair
follicle formation and low levels stimulating the differentiation
of IFE and sebocytes (10, 114.
A second important signaling pathway in the cutaneous
epithelium involves the secreted protein Sonic hedgehog
(SHH), its receptors Patched (PTCH/Ptch) and Smoothened,
and downstream transcription factors of the GLI family (124.
Hair follicle development in SHH null mutant mice arrests
after the initial epidermal-dermal interactions, indicating that
SHH signaling is required for normal advancement beyond the
hair germ stage of development (13, 144. Mice that are
homozygous null for Indian hedgehog (IHH) have major
skeletal defects and die at birth, but no analysis of their skin
has been described (154. GLI2 is the key mediator of SHH
responses in skin and positively regulates GLI1; GLI2 knock-
out mice show a similar arrest in hair follicle development to
SHH null animals (164. In adult mouse skin SHH is required
for the normal hair growth cycle (17), and treatment with
antibodies to SHH blocks the growth of anagen hair follicles
(18~. In both the knockouts and the antibody-treated epider-
mis some markers of follicular differentiation are expressed,
and it seems that SHH is primarily required for growth rather
than differentiation (16, 19, 20~.
Both the ,B-catenin and hedgehog (Hh) pathways play a role in
epidermal carcinogenesis. PTCH is mutated in human nevoid BCC
syndrome (Gorlin-Goltz's syndrome), a hereditary predisposition
to BCCs, medulloblastomas, and rhabdomyosarcomas (21-24~.
PTCH/Ptch mutation results in overexpression and activation of
GLI1, and there is evidence that activation of GLI1 and GLI2 leads
to the development of BCCs (25, 26~. Constitutive activation of
SHH signaling in transgenic mouse and human skin also leads to the
formation of BCCs (27-29~. Smoothened is required for activating
transcription of Hh target genes, and overexpression of constitu-
tively active Smoothened results in tumors similar to those caused
by SHH overexpression (30~.
Transgenic mice that overexpress N-terminally truncated
13-catenin in the basal layer of the IFE and hair follicle develop
hair tumors (trichofolliculomas and pilomatricomas), and acti-
vating mutations in [3-catenin have been found at high frequency
in human pilomatricomas (7, 31~. Activation of `~-catenin sig-
This paper results from the Arthur M. Sackier Colioquium of the Nationai Acaclemy of
Sciences, "Regenerative Meclicine," heicl October 18-22, 2002, at the Arnoicl ancl Mabe!
Beckman Center of the Nationai Academies of Science ancl Engineering in frvine, CA.
Abbreviations: Hh, hecigehog; DHH, Desert Hh; ~HH, Inclian Hh; SHH, Sonic Hh; PTCH/Ptch,
Patchecl; IFE, interfoilicuiar epiclermis; BCC, basal ceil carcinoma.
*C.N. ancl A.B.U. contributecl equally to this work.
~To whom corresponclence shouicl be adciressed. E-maii: fiona.wattC?cancer.org.uk.
C) 2003 by The Nationai Acaclemy of Sciences of the USA
PNAS | Seplember 30, 2003 | vo~. 100 | suppl. ~ | 11873-~1SBO
OCR for page 58
naling is associated with cancer in many different tissues,
whereas inhibition is not thought to play any role in the disease
(32~. It is thus surprising that mice expressing N-terminally
truncated Left (l`NLefl), which blocks ,B-catenin signaling in the
basal layer of the IFE and hair follicle (K147\NLefl transgenics),
develop spontaneous tumors (10~. Consistent with the role of
f-catenin levels in controlling lineage selection, the tumors in
K14l\NLefl transgenics show sebaceous and squamous differ-
entiation rather than hair follicle differentiation (104.
There is good evidence for crosstalk and conservation in Hh
and Wnt signaling. Both pathways share components such as
GSK, CK1, and the F box protein Slimb (33, 34) and involve G
protein-coupled receptors (Smoothened in the Hh pathway and
Frizzled receptors for Wnts). When ,B-catenin is overexpressed
in the epidermis, SHH transcription is elevated (7~. Conversely,
SHH expression is inhibited in ,l3-catenin null epidermis (8), and
overexpression of the Wnt antagonist Dickkopfl blocks SHH
expression (35~. However, SHH is not simply downstream of Wnt
signaling because WntSa is a target of SHH in hair follicle
morphogenesis (6) and Ptch mRNA is up-regulated in the
spontaneous tumors of K14ANLefl transgenic mice (10~. In
addition, Wnt expression is altered by activation of SHH in
human BCCs (36~.
To investigate the mechanism leading to development of
spontaneous tumors in K147\NLefl transgenic animals we ana-
lyzed components of the Hh signaling pathway in more detail.
Whereas SHH has been shown to be important for hair follicle
development and BCC formation, our results indicate that IHH
is involved in growth and differentiation of sebocytes in normal
skin and in the formation of sebaceous tumors of human and
mice. We propose that i\NLefl and IHH cooperate to control
proliferation and differentiation of sebocyte progenitors.
Materials and Methods
Source of Tissues. Spontaneous skin tumors from K147\NLefl
transgenic mice (10) were formalin-fixed and paraffin-
embedded. Formalin-fixed and paraffin-embedded human skin
tumors (nine sebaceous adenomas and one pilomatricoma) were
obtained from Beth Israel Deaconess Medical Center. Sections
from normal mouse and human skin were obtained from the
Center for Nutrition and Toxicology, Karolinska Institute,
Stockholm and the Department of Dermatology, Karolinska
Hospital, Stockholm, respectively.
Immunohistochemical Staining of Tumors and Normal Skin. Paraffin
sections were dewaxed, rehydrated, and incubated in 1% H2O2
in methanol for 30 min. The sections were then treated with 10
mM citrate buffer at 97°C for 30 min. Sections were incubated
overnight at 4°C with diluted rabbit polyclonal antibodies di-
rected against human PTCH1 (Research Genetics, Huntsville,
AL, and Santa Cruz Biotechnology, sc-6149, both 1:200), GLI1
(AbCam, Cambridge, U.K., 1:300), GLI2 (Research Genetics,
1:50), and pan-HH (Santa Cruz Biotechnology, sc-9024,1:100) or
goat polyclonal antibodies directed against human SHH (Santa
Cruz Biotechnology, sc-1194, 1:100), IHH (Santa Cruz Biotech-
nology, sc-1196, 1:30), and Desert Hh (DHH) (Santa Cruz
Biotechnology, sc-1193, 1:100~. All antibodies were diluted in
PBS containing 0.1% BSA. Detection was carried out with the
Vectastain Elite Kit (Vector Laboratories) by using rabbit or
goat IgG. After washing the sections with PBS, biotinylated
secondary antibodies were added for 30 min at room tempera-
ture. After extensive rinsing and incubation with avidin-biotin,
immunoperoxidase antibody staining was visualized with 3-ami-
no-9-ethylcarbazole (Vector Laboratories), and sections were
counterstained with Mayer's hematoxylin. As controls primary
antibodies were omitted (in the case of PTCH1 N terminus,
IHH, DHH, and pan-Hh antibodies) or incubated with the
11874 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 1834202100
corresponding peptide immunogen (PTCH C terminus, SHH,
GLI1, and GLI2 antibodies) during the staining procedure.
Sebocyte Culture and Retroviral Infection. SZ95 are a line of human
facial skin sebocytes that have been immortalized by transfection
of simian virus 40 large T antigen (37) and were cultured in
Sebomed medium (Biochrom, Berlin) containing lO~o FCS
(Sera-Lab, Crawley Down, Sussex, U.K.), 3 ng/ml keratinocyte
growth factor (PeproTech, Rocky Hill, NJ), and 20 ng/ml
epidermal growth factor (PeproTech). SZ95 cells were retrovi-
rally infected by using supernatant of AM12 amphotropic pro-
ducer cells as described (38) and selected in 1.5 ,ug/ml puromy-
cin. To inhibit Hh signaling, subconfluent and undifferentiated
SZ95 cultures were treated with 5 ,uM cyclopamine in methanol;
control cells were treated with methanol alone.
To generate growth curves, the Cyto Tox assay kit (Promega)
was used to determine total cell number. A total of 2 x 103 SZ95
sebocytes were seeded per well into 96-well plates, and the
number of cells per well was determined in triplicate at different
time points for up to 9 days of cell culture.
Staining of SZ95 Cells and Epidermal Whole Mounts. SZ95 sebocytes
grown on coverslips were fixed in 4% paraformaldehyde and
subjected to indirect immunofluorescence staining as described
(38~. Primary antibodies used were anti-PTCH antibody (C
terminus, Research Genetics, 1:200), anti-IHH antibody (Santa
Cruz Biotechnology, 1:200), anti-pan-Hh antibody (Santa Cruz
Biotechnology, sc-9024, 1:200), anti-GLI1 antibody (AbCam,
1:300), anti-GLI2 antibody (Research Genetics, 1:50), FWCAD
(rabbit anti-E-cadherin antibody; ref. 39), 9E10 (anti-Myc-tag
antibody, 1:400), and 12CA5 (antihemagglutinin tag; ref. 40~.
Secondary antibodies were conjugated with AlexaFluor 488 or
AlexaFluor 594 (Molecular Probes). In some experiments cells
were counterstained with 4',6-diamidino-2-phenylindole (Mo-
lecular Probes) to identify nuclei. To stain lipids, SZ95 sebocytes
were incubated with Nile red (0.1 ,ug/ml in PBS, Sigma) for 30
min at room temperature, then washed several times with PBS
and distilled water.
Whole mounts of tail epidermis were prepared from WT and
K141\NLefl transgenic mice bred in a CBAxC57Bl6 F~ mixed
background (10) by using a recently developed method (70~. The
tail was slit length-wise, and the skin was peeled from the tail.
The skin was then incubated in EDTA until the epidermis could
be peeled as an intact sheet from the dermis. The epidermis was
fixed in 4% formal saline (Sigma) for 2 h at room temperature,
then blocked and permeabilized in phosphate buffer consisting
of 0.5% skim milk powder, 0.25% fish skin gelatin (Sigma), and
0.5% Triton X-100 in TBS (0.9% NaCl/20 mM Hepes, pH 7.2~.
Epidermal sheets were incubated with primary antibodies di-
luted in phosphate buffer overnight at room temperature with
gentle agitation. Primary antibodies used were anti-GLI1 anti-
body (AbCam, 1:300), anti-GLI2 antibody (Research Genetics,
1:50), and anti-,8~ integrin antibody (MB 1.2, kindly provided by
B. Chan, University of Western Ontario, London, Canada; ref.
414. The epidermis was then washed for >4 h with PBS
containing 0.2% Tween 20, changing the buffer several times.
Incubation steps with secondary antibodies were performed in
the same way, and epidermal sheets were rinsed in distilled water
before mounting in Gelvatol (Monsanto, St. Louis) containing
2.5% 1,4-diazabicyclot2.2.2.Joctane (Sigma).
Images of SZ95 sebocytes and epidermal whole mounts were
acquired by using a Zeiss 510 confocal microscope. Scans of
whole mounts are presented as z-projections of ~30 optical
sections captured.
Western Blotting. Protein lysates for Western blotting were ex-
tracted from undifferentiated, differentiated, and retrovirally
infected cultures of SZ95 sebocytes. Cells were washed twice
Niemann eta/.
OCR for page 59
Table 1. Expression of components in the SHH-Ptch signaling
pathway in spontaneous sebaceous skin tumors of K14ANLef1
transgenic mice
PTCH1
C N
No. Tumor terminal terminal SHH IHH DHH pan-HH GLI1 GLI2
1 Pap ++
2 Seb +
3 SA +
4 Seb +
5 SA
6 SA
7 SA
8 SA
9 SA
10 SA
11 Pap
12 Seb
++
++
++
++
++
+ + ++ _
+ + ++ _
+ — ++ _
+ — ++ _
+ + ++ _
+ + ++ _
+ + ++ _
+ — ++ _
+ — ++ _
+ + ++ _
+ + ++ _
+ — ++ _
+ + +
++ + ++
+ + ++
++ + +
++ + +
++ + ++
+ + ++
++ + ++
++ + ++
+ + +-
++ + ++
+ + +
pap, squamous papilloma with sebocyte differentiation; seb, sebeoma; SA,
sebaceous adenoma. Expression strength was scored as follows: -, no detectable
specific immunoreactivity; +, specific immunoreactivity in a moderate number of
cells; + +, strong and specific immunoreactivity in a high number of cells.
with ice-cold PBS and TNE buffer containing 40 mM Tris HCl
(pH 7.4), 10 mM EDTA, and 150 mM NaCl was added to harvest
the cells. SZ95 sebocytes were washed with ice-cold PBS and
lysed in 200 ,ul WCE buffer containing 20 mM Hepes (pH 7.9),
0.42 M NaCl, 0.5% Nonidet P-40, 0.5% deoxycholic acid, 25%
glycerol, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DTT, and pro-
tease inhibitors. Protein lysates were incubated at 4°C for 30 min
with constant agitation and centrifuged at 4°C for 30 min at
14,000 rpm. Proteins in the supernatant were separated on 7.5%
(PTCH) and 15% (Hh) SDS/PAGE gels, electroblotted, and
probed with antibodies against PTCH (C terminus, 1:200 dilu-
tion, Research Genetics), pan-Hh (Santa Cruz Biotechnology,
1:250), Myc-epitope (9E10, Santa Cruz Biotechnology), and
hemagglutinin tag (CAS12; ref. 40) as described (38~. Immuno-
reactive proteins were visualized by chemiluminescence (ECL,
Amersham Pharmacia). Blots were stripped with glycine buffer
and reprobed with an antiactin antibody (AC-40, Sigma, 1;2000)
as a control for equal loading.
Results
IHH Is Expressed in Mouse and Human Sebaceous Tumors. Transgenic
mice (K142\NLefl) expressing a truncated mutant form of the
transcription factor Lefl (lacking the first 32 aa that contain the
I3-catenin binding domain) under control of the keratin 14
promoter develop spontaneous skin tumors (10~. The majority of
the tumors are sebaceous adenomas, consisting of differentiated
sebocytes surrounded by undifferentiated progenitor cells. The
second most common tumor type contains areas of sebaceous
differentiation and areas of squamous differentiation or undif-
ferentiated basal keratinocytes (sebeomas; ref. 10~. Some of the
mice develop squamous papillomas, but even though IFE dif-
ferentiation predominates in these tumors there are also clusters
of sebocytes.
We showed previously by in situ hybridization that ptch RNA
was up-regulated in all of the tumors of K142\NLefl mice (10~.
To investigate the underlying mechanism, we analyzed expres-
sion of various components of the Hh signaling cascade in more
detail. We performed immunohistochemical staining with anti-
bodies against PTCH, its three known ligands (SHH, DHH, and
IHH), and antibodies to downstream transcription factors of the
GLI family, GLI1 and GLI2, that are known to be expressed in
the epidermis (ref. 16 and Table 1~. We examined 12 tumors
Niemann et a/.
Fig. 1. Expression of proteins in the PTCH-SHH signaling pathway in
K14ANLef1 transgenic mouse sebaceous tumors. Immunostaining was per-
formed with antibodies to the proteins indicated. (a) Ptch1 C terminus. (b)
Ptch1 N terminus. (c) SHH. (d) IHH. (e) DHH. (fl pan-HH. (g) GLI1 (h) GLI2. (Scale
bar: 200 ,um.)
from K141\NLefl mice, comprising sebaceous adenomas, sebeo-
mas, and squamous papillomas with sebocyte differentiation
(Fig. 1 and Table 1~.
Staining with antibodies to the N- and C-terminal regions of
Ptchl established that the protein was up-regulated. The Ptchl
C-terminal antibody showed prominent immunoreactivity in
both the differentiated sebocytes and the surrounding basaloid
cells, labeling the cytoplasm, nucleus, and cell membrane (Fig.
la). In contrast, the Ptchl N-terminal antibody stained the
cytoplasm and plasma membrane, but gave no or weak nuclear
staining (Fig. lb). These observations allow us to draw two
conclusions: first, that the elevated ptch mRNA in the tumors
(10) correlates with elevated PTCH protein; second, that Ptchl
is unlikely to be mutated, because the majority of PTCH
mutations in tumors result in premature termination of Ptch
translation leading to the absence of the C terminus (424.
The expression of SHH protein was generally weak in spon-
taneous mouse sebaceous tumors. The strongest immunoreac-
tivity with the SHH antibody was seen in the basaloid progenitor
cells with only faint staining in the mature sebocytes (Fig. lc and
Table 1~. Immunohistochemical staining with the DHH antibody
PNAS | September 30, 2003 | vol. 300 | suppl. ~ | 11875
OCR for page 60
Table 2. Expression of components in the SHH-PTCH signaling
pathway in sebaceous human skin tumors
PTCH 1
C N
No. Tumor terminal terminal SHH IHH DHH pan-HH GLI1 GLI2
1 SA ++ + + ++ - ++ nd +
2 SA ++ + - ++ - ++ nd +
3 SA ++ + + ++ + ++ nd
4 SA ++ + - ++ - ++ nd +
5 SA + + + + + + +
6 SA ++ + + ++ - ++ ++ +
7 SA ++ + + ++ - ++ ++ +
8 SA ++ + + ++ - ++ ++ +
9 SA + + + + + + _ +
SA, sebaceous adenoma; nd, not determined. Expression strength was
scored as follows: -, no detectable specific immunoreactivity; +, specific
immunoreactivity in a moderate number of cells; ++, strong and specific
immunoreactivity in a high number of cells.
was negative in both the undifferentiated and differentiated cells
of the tumors (Fig. le and Table 1~. In contrast, IHH protein was
strongly expressed in the mature sebocytes of the tumors,
whereas the basaloid progenitor cells were completely negative
in all cases (Fig. Id and Table 1~. Staining with the pan-Hh
antibody was seen both in basaloid and sebaceous gland cells and
corresponded to the combination of the staining pattern for
SHH and IHH in the skin tumors (Fig. if). GLI1 and GLI2
proteins were expressed in the basaloid cells and in the sebocytes
of the spontaneous skin tumors of K141\NLefl mice, although
they were absent from the most highly differentiated sebocytes
in the centers of the tumors (Fig. 1 g and h and Table 1~. We
conclude that the major Ptch ligand expressed in the sebaceous
tumors is IHH and that Hh signaling is active because Ptchl is
expressed. The nuclear localization of GLI1 is a further indica-
tion that Hh signaling is activated in the sebaceous tumors.
We next examined a panel of nine human sebaceous adeno-
mas. The immunohistochemical staining of components in the
Hh-PTCH1 signaling pathwaywas similar to that observed in the
spontaneous mouse tumors (Table 2 and Fig. 24. All of the
human tumors stained positive with antibodies to the N and C
termini of PTCH1 (Fig. 2 a and b). Strong staining with
antibodies against IHH was detected in the mature sebocytes of
the human tumors (Fig. 2d), whereas DHH protein was either
not detectable (Fig. 2e) or only weakly positive (Table 2) and
SHH staining was weak (Fig. 2c). Staining with the pan-He
antibody corresponded to the combined staining patterns for the
individual Hh proteins (Fig. 2f). GLI1 and GLI2 were located
in the nucleus and in the cytoplasm (Fig. 2 g and h), although
GLI1 antibodies stained the cytoplasm more strongly than the
nucleus (Fig. 2g).
Taken together, our results show that components of the
Hh-PTCH1 signaling pathway are up-regulated in sebaceous
skin tumors of human and mouse, suggesting that the pathway is
active in the tumor cells. IHH is the most prominent ligand of the
Hh pathway in sebaceous tumors. In contrast, IHH protein was
not detectable in human pilomatricomas, which are hair follicle
tumors (Fig. 3b).
IHH and GLI1 Expression in Normal Epidermis. To investigate whether
the strong expression of IHH in sebaceous tumors was indicative
of normal sebaceous differentiation, we analyzed expression of
IHH, GLI1, and GLI2 protein in normal skin (Fig. 3~. In both
human and mouse skin, IHH protein was strongly expressed in
the differentiated sebocytes of sebaceous glands and undetect-
able in the IFE or the hair follicles (Fig. 3a and data not shown).
~1876 1 www.pnas.org/cgi/doi/ ~ 0. ~ 073/pnas. ~ 834202 ~ Oo
Fig. 2. Expression of proteins in the SHH-PTCH signaling pathway in human
sebaceous tumors. Immunostaining was performed with antibodies to the
proteins indicated. (a) PTCH1 C terminus. (b) PTCH1 N terminus. (c) SHH. (d)
IHH. (e) DHH. (fl pan-HH. (g) GLI1. (h) GLI2. (Scale bar: 200 ,um.)
To examine the expression of GLI1 and GLI2 we stained
whole mounts of mouse tail skin (70~. We observed weak
cytoplasmic immunostaining with antibodies against GLI1 in the
outer root sheath of the hair follicle and in the IFE, consistent
with a previous study (43~. In contrast, strong staining with the
anti-GLI1 antibody was seen in the nuclei of progenitor cells in
the sebaceous gland (Fig. 3c, arrowheads). Furthermore, we
observed nuclear GLI1 protein in sebocyte progenitors that
formed ectopically along the deformed hair follicle structures in
K14/\NLefl transgenic mice (Fig. 3d, arrowheads, and ref. 10~.
GLI2 was strongly expressed in the cytoplasm of cells in the
permanent portion of the follicle below the sebaceous glands (a
region where the follicle stem cells are concentrated; ref. 70) and
had a nuclear distribution in some cells at the periphery of the
sebaceous glands (Fig. 3e, arrowheads). In K14~\NLefl epider-
mis staining for GLI2 was uniform along the deformed follicles
and was predominantly cytoplasmic with some weak nuclear
staining (Fig. 3f). Taken together, our results suggest that IHH
and GLI1 are expressed in normal sebocytes, the ectopic sebo-
cytes induced by i\NLefl, and sebaceous skin tumors of mouse
and human. The distribution of GLI2 in normal follicles is
consistent with its primary function in SHH signaling (16).
Niemann et a/.
OCR for page 61
a
a. ~~ ~
~ ~ . ~
Hi.
Fig. 3. Expression of IHH, Gli1, and Gli2 proteins in normal and K14/\NLef1
transgenic mouse skin and in human pilomatricoma. (a) Immunostaining with
IHH antibody in normal mouse skin. Note strong reactivity in the sebaceous
glands (arrowheads). (b) In human pilomatricoma (a hair follicle-derived
tumor) Immunostaining for IHH was not detected. (c-f) Immunostaining for
GLI1 (green, c and d) and GLI2 (green, e and f) proteins in whole mounts of tail
epidermis of WT (c and e) and K147\NLef1 transgenic (d and f) mice. Note
strong expression of GLI 1 in the nuclei of cells in the normal sebaceous glands
(arrowheads in c) and newly differentiating sebocytes along the deformed
transgenic hair follicles (arrowheads in d). Note strong and predominantly
cytoplasmic staining of GLI2 in the permanent portion of the follicle belowthe
sebaceous glands in WT epidermis (e, brackets) and weaker, more uniform
staining in transgenic epidermis (f, bracket indicates equivalent region to
those shown in e). Note weak nuclear staining for GLI2 in normal sebaceous
glands (e, arrowheads). Whole mounts were double-labeled for,B1 integrins
(red) to reveal the basal IFE, periphery of sebaceous glands and outer root
sheath of the hair follicles. [Scale bars: 100 Am (a and b) and 50 ,um (c-f).l
IHH Regulates Growth of Human Sebocytes in Vitro. To investigate
the role of IHH signaling in sebocyte differentiation we used the
SZ95 line of human facial skin sebocytes that have been immor-
talized by transfection of simian virus 40 large T antigen (37~.
SZ95 sebocytes can be induced to differentiate into mature
sebocytes by growing the cells to high density (374. Nile red is a
hydrophobic dye that is highly selective for lipids and can
therefore be used as a marker for differentiated mature sebo-
cytes (ref. 37 and Fig. 4 a and b).
First, we compared expression of IHH and PTCH1 in undif-
ferentiated and differentiated SZ95 sebocytes by immunofluo-
rescense staining. IHH was only weakly expressed in undiffer-
entiated cells (Fig. 4c). In contrast, IHH could be readily
detected in the cytoplasm in close approximation to the plasma
membrane of differentiated mature sebocytes (Fig. 4d ). A
Niemann et a/.
undifferentiated differentiated
J undif. dif.
4~= PTCH | _ ~ Hh
actin :~ t
acid
_.
_
_—
_I
ev ANLef1
_
_ Hh
__ PIT
AH
_ actin
Fig. 4. Expression of IHH, PTCH1, GLI1, and GLI2 in undifferentiated and
differentiated human SZ95 sebocytes in vitro. (a, c, and e) Undifferentiated
cells. (b, d, and f) Postconfluent, differentiated cells. (g-/) Partially differen-
tiated cultures. (a and b) Nile red staining of lipids. (b) Note dramatic increase
in the number of lipid droplets in differentiated SZ95 sebocytes (arrows). (c
and d) Immunostaining for IHH protein (green) and E-cadherin (to reveal
cell-cell borders; red). (d) IHH protein levels are up-regulated in differentiated
sebocytes (arrows). (e and f) Immunostaining for PTCH1 protein (green) with
4',6-diamidino-2-phenylindole nuclear counterstain (blue). PTCH 1 staining is
up-regulated in the cytoplasm of differentiated sebocytes (arrows in f).
Immunostaining for GLI1 (g and h) and GLI2 (] (green) with 4',6-diamidino-
2-phenylindole nuclear counterstain (blue), showing nuclear and cytoplasmic
GLI localization in areas where the cultures are still undifferentiated (g and hi
and exclusively cytoplasmic localization in suprabasal, terminally differenti-
ating sebocytes (h and i Inset, arrows). [Scale bar: 20 Am (a-/~.] (I) Western blots
of protein Iysates prepared from undifferentiated and differentiated SZ95
sebocytes (Left and Center) and retrovirally infected differentiated cells
(Right) probed with antibodies against PTCH1 C terminus (160 kDa), pan-Hh
(processed form of ~25 kDa) and, as a loading control, actin (42 kDa). ev,
empty vector control; ANLef1, retroviral vector.
similar staining pattern was observed with anti-PTCH1 antibod-
ies: weak expression in undifferentiated cells (Fig. 4e) and
up-regulated levels in differentiated sebocytes (Fig. 4f ). PTCH1
was detected only in the cytoplasm and not in the nucleus of the
sebocytes (Fig. 4f). The immunofluorescence results were con-
firmed by Western blotting with antibodies to PTCH1 and a
pan-Hh antibody: levels of both PTCH1 and Hh were higher in
differentiated SZ95 cells than in undifferentiated cells (Fig. 4j
Left and Center). These data indicate that ITCH and its receptor,
PTCH1, are up-regulated during sebocyte differentiation in
vitro.
The observation that IHH was expressed in mature sebocytes
and that GLI1 was activated in sebocyte progenitors suggested
a paracrine effect of IHH. This finding was supported by
examining GLI1 and GLI2 expression in SZ9S sebocytes (Fig. 4
Hi). In newly confluent cultures both transcription factors were
PNAS | September 30, 2003 | volt. 100 | suppl. ~ | 11877
OCR for page 62
20000
1 5000
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8
E 10000
a
soon
/
. ~ ~ + ~ MnOH
ol
4 5
80000
60000
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-
o
20000
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T2
~ dnLef1
3 5 7 9
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~ stem cell
unlead watt ~ANLef1
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sel'~eyte hair follicle
Fig. 5. Manipulating Hh and ,B-catenin signaling regulates growth and differentiation of human sebocytes in vitro. (a-c) Inhibition of Hh signaling in human
sebocytes decreases proliferation and stimulates differentiation. (a) Growth curve of SZ95 cells treated with Hh inhibitor cyclopamine or solvent methanol. (b)
Treatment of undifferentiated SZ95 cells with methanol did not affect differentiation as evaluated by Nile red staining, whereas treatment with cyclopamine
(c) resulted in accumulation of Nile red-positive lipid droplets (arrows). (d-J) Retroviral transduction of SZ95 cells with pBabepuro (d and g), pBabei\NLef1 (e and
h), and /\N,B-catenin/T2 (f and /~. Transduced gene products were detected by immunostaining with anti-Myc tag (e) and antihemagglutinin tag (d and f). Cells
were labeled with antibodiesspecificfor E-cadherin (to reveal cell-cell borders; red, a-/) and IHH (green, q-/~. /\NLef1 increased production of IHH protein (arrows
in h). [Scale bars: 20 Am (b-i).] (I) Growth curve of cells transduced with pBabepuro (ev), pBabe~\NLef1 (dnLef1), or i\N,B-catenin (T2). (k) Model of interactions
between ,B-catenin and Hh signaling in epidermis. SHH promotes proliferation of progenitor cells of the hair lineage, whereas IHH stimulates proliferation of
sebocyte precursors. The IHH signal is proposed to be produced by differentiated sebocytes and act on the sebocyte progenitors in a paracrine fashion.
found in the nucleus and cytoplasm of cells that had not yet
accumulated substantial numbers of lipid droplets (Fig. 4 g and
i). Suprabasal and differentiated cells had cytoplasmic but not
nuclear GLI1 (Fig. 4h, arrows) and GLI2 (Fig. 4i Inset, arrow).
To investigate the role of IHH signaling in growth and
differentiation of human sebocytes in vitro, we treated SZ95
sebocytes with cyclopamine, a specific and potent inhibitor of Hh
signaling (44-46~. Treatment of SZ95 sebocytes with 5 AM
cyclopamine decreased proliferation (Fig. 5a) and stimulated
differentiation, as evaluated by Nile red staining (Fig. 5 b and c).
From this we conclude that IHH promotes the proliferation of
undifferentiated sebocytes.
~ ~ 878 1 www pnas org/cgi/doi/ ~ 0. ~ 073/pnas. ~ 834202 ~ 00
,6-Catenin Signaling Regulates Growth and IHH Expression of Human
Sebocytes in Vitro. To investigate whether I3-catenin signaling
affected the growth and differentiation of sebocytes in vitro we
infected SZ95 sebocytes with retroviral expression vectors for
mutant proteins of the ,B-catenin signaling cascade. SZ95 sebo-
cytes were infected with pBabeT2, a stabilized, N-terminally
deleted 13-catenin mutant (~\N13-catenin) (38), pBabe/\NLefl to
block ,B-catenin signaling (10) and the empty vector pBabepuro,
as a control. Expression of the mutant proteins was analyzed by
immunofluorescence staining with antibodies against the T2
hemagglutinin tag (Fig. 5 d end f) and the i\NLefl myc-tag (Fig.
Se), and by Western blotting (data not shown). As expected,
Niemann et a/.
OCR for page 63
i\NLefl could be readily detected in the nuclei of infected SZ95
cells (Fig. 5e), whereas i\Nf3-catenin (T2) was expressed in the
cytoplasm, nucleus, and plasma membrane of infected cells (Fig.
5f). Cells expressing the empty vector (Fig. 5d) served as a
negative control.
We analyzed whether manipulating ,l3-catenin signaling in
infected SZ95 sebocytes could regulate IHH synthesis. ANLefl
increased expression of IHH in SZ95 sebocytes compared with
the empty vector control cells whereas i\NI3-catenin slightly
decreased expression of IHH (Figs. 4j Right and 5 g-i). Neither
i\NLefl nor /\N!3-catenin had any effect on sebocyte differen-
tiation as evaluated by Nile red staining of low-density cultures
(data not shown). i\NLefl stimulated proliferation of undiffer-
entiated sebocytes compared with control cells (Fig. 5j). This
finding is in contrast to the effects of the constructs on the
growth of normal human IFE keratinocytes: i\NLefl decreases
proliferation and i\N,l3-catenin stimulates proliferation of puta-
tive IFE stem cells in vitro (ref. 38 and data not shown). The
stimulation of SZ95 cell proliferation by '\NLefl was abolished
by cyclopamine (data not shown). Our results demonstrate that
expression of i\NLefl increases IHH expression in differentiated
sebocytes and stimulates proliferation of undifferentiated
sebocytes.
Discussion
There is increasing evidence that Hh signaling can regulate
growth and survival in both differentiated and undifferentiated
cells (47-49) and can also regulate lineage-specific differentia-
tion (50-524. Hh family members tend to act in a paracrine
fashion (12~.
We have shown that IHH is up-regulated in the differentiated
sebocytes of normal human and mouse epidermis and in seba-
ceous tumors. The nuclear accumulation of GLI1 in undiffer-
entiated sebocytes in vivo is an indication of active Hh signaling
(53-56~. The negative effect of cyclopamine on proliferation of
undifferentiated sebocytes in vitro is direct evidence that IHH
stimulates proliferation of sebaceous progenitor cells. Thus we
propose that, whereas SHH promotes proliferation of progeni-
tors of the hair lineages (20), IHH produced by mature sebocytes
promotes proliferation of sebaceous progenitors in a paracrine
manner (Fig. 5k). Whereas f-catenin levels regulate lineage
choice within the epidermis (1), Hh promotes the proliferation
of committed progenitors (Fig. 5k). SHH and IHH appear to
signal via common receptors, and so specificity would be at the
level of the cell type expressing each ligand (124.
We observed that ANLefl increased IHH expression in cul-
tured sebocytes, and we propose that this is one mechanism by
which i\NLefl stimulates proliferation of sebocyte progenitors
(Fig. 5k). The formation of spontaneous sebaceous tumors in
K14l\NLefl mice is likely to be a consequence of the increased
number of sebocyte progenitors, although whether, by analogy
1. Niemann, C. & Watt, F. M. (2002) Trends Cell Biol. 12, 185-192.
2. Fuchs, E. & Raghavan, S. (2002) Nat. Rev. Genet. 3, 199-209.
3. Millar, S. E. (2002) J. Invest. Dermatol. 118, 216-225.
4. Owens, D. M. & Watt, F. M. (2003) Nat. Rev. Cancer 3, 444-451.
5. DasGupta, R. & Fuchs, E. (1999) Development (Cambridge, U.K.) 126, 4557-
4568.
6. Reddy, S., Andl, T., Bagasra, A., Lu, M. M., Epstein, D. J., Morrisey, E. E. &
Millar, S. E. (2001) Mech. Dev. 107, 69-82.
7. Gat, U., Dasgupta, R., Degenstein, L. & Fuchs, E. (1998) Cell 95, 605-614.
8. Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G. & Birchmeier, W. (2001)
Cell 105, 533-545.
9. Merrill, B. J., Gat, U., DasGupta, R. & Fuchs, E. (2001) Genes Dev. 15,
1688-1705.
10. Niemann, C., Owens, D. M., Hulsken, J., Birchmeier, W. & Watt, F. M. (2002)
Development (Cambridge, U.K) 129, 95-109.
11. DasGupta, R., Rhee, H. & Fuchs, E. (2002) J. Cell Biol. 158, 331-344.
12. Ingham, P. W. & McMahon, A. P. (2001) Genes Dev. 15, 3059-3087.
Niemann et a/.
with the effects of overexpressing SHH (27-30), IHH overex-
pression is sufficient to induce the tumors remains to be tested.
One mechanism by which SHH stimulates proliferation is by
up-regulation of D-type cyclins (16, 57, 58~. However,
K14~\NLefl transgenic mouse tumors do not express cyclin D1,
because 13-catenin signaling is inhibited (10~. An alternative
mechanism by which IHH could drive sebaceous proliferation in
the presence of i\NLefl would be by opposing Wafl /p21-
mediated growth arrest (59~.
Sebaceous tumors are a characteristic feature of Muir-Torre
syndrome in which patients develop colon cancer (604. Muir-
Torre patients and other individuals with colon cancer (sporadic
and hereditary nonpolyposis colon cancer) have microsatellite
instability. Interestingly, microsatellite instability in colon can-
cers can result in frameshift mutations in the TCF4 gene (61~.
These observat~ons raise the intriguing possibility that sebaceous
tumors of Muir-Torre patients might have frameshift mutations
in the Lefl gene.
Although IHH is linked primarily to cartilage differentiation
(15) there are several reports that it plays an important role in
epithelia. Furthermore, in both cartilage and epithelia there is an
association between IHH and hormonal regulation. In mouse
uterus, IHH mediates epithelial-mesenchymal interactions and
is regulated by progesterone (62, 63~. IHH is also expressed in
mammary gland epithelium and is up-regulated during preg-
nancy and lactation (64~. Our observation of IHH expression in
sebocytes, which are under the control of androgens and a range
of other hormones (65, 66), fits very well with these observations.
It is also of interest that liganded androgen receptor represses
,B-catenin/T cell factor-mediated transcription (67-69), suggest-
ing a possible synergy between l\NLefl and androgens in
promoting sebocyte differentiation in addition to the potential
synergy between androgens and IHH in stimulating proliferation
of sebocyte progenitors.
Taken together, our data suggest that IHH and i\NLefl
function in concert to change the proliferative and differen-
tiation program of epidermal stem cell daughters. We are far
from understanding the detailed interactions between Wnt
and Wh signaling cascades in the epidermis and how they, in
turn, interact with the numerous other signaling pathways that
control epidermal stem cell fate (1, 2~. The recent find~ng that
in transgenic mouse epidermis the same f-catenin mutation
exerts different effects depending on the cells in which it is
expressed underlines the importance of cellular context and
microenvironment in the control of tissue renewal and differ-
entiation (114.
We are grateful to Angela Mowbray and the Cancer Research UK
Histopathology Unit for expert technical assistance. This work was
supported by Cancer Research UK (C.N. and F.M.W.) the European
Union (F.M.W.), and the Swedish Cancer Fund (A.B.U. and R.T.~.
13. St-Jacques, B., Dassule, H. R., Karavanova, I., Botchkarev, V. A., Li, J.,
Danielian, P. S., McMahon, J. A., Lewis, P. M., Paus, R. & McMahon, A. P.
(1998) Curr. Biol. 8, 1058-1068.
14. Chiang, C., Swan, R. Z., Grachtchouk, M., Bolinger, M., Litingtung, Y.,
Robertson, E. K., Cooper, M. K., Gaffield, W., Westphal, H., Beachy, P. A. &
Dlugosz, A. A. (1999) Dev. Biol. 205, 1-9.
15. St-Jacques, B., Hammerschmidt, M. & McMahon, A. P. (1999) Genes Dev. 13,
2072-2086.
16. Mill, P., Mo, R., Fu, H., Grachtchouk, M., Kim, P. C., Dlugosz, A. A. & Hui,
C. C. (2003) Genes Dev. 17, 282-294.
17. Sato, N., Leopold, P. L. & Crystal, R. G. (1999) J. Clin. Invest. 104,
855-864.
18. Wang, L. C., Liu, Z. Y., Gambardella, L., Delacour, A., Shapiro, R., Yang, J.,
Sizing, I., Rayhorn, P., Garber, E. A., Benjamin, C. D., ef al. (2000) J. Invest.
Dermatol. 114, 901-908.
19. Oro, A. E. & Scott, M. P. (1998) Cell 95, 575-578.
20. Callahan, C. A. & Oro, A. E. (2001) Curr. Opin. Genet. Dev. 11, 541-546.
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11879
OCR for page 64
21. Hahn, H., Wicking, C., Zaphiropoulous, P. G., Gailani, M. R., Shanley, S.,
Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A. B., Gillies, S., et
al. (1996) Cell 85, 841-851.
22. Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J. W., Bonifas,
J. M., Quinn, A. G., Myers, R. M., Cox, D. R., Epstein, E. H., Jr., & Scott, M. P.
(1996) Science 272, 1668-1671.
23. Goodrich, L. V., Milenkovic, L., Higgins, K. M. & Scott, M. P. (1997) Science
277, 1109-1113.
24. Hahn, H., Wojnowski, L., Zimmer, A. M., Hall, J., Miller, G. & Zimmer, A.
(1998) Nat. Med. 4, 619-622.
25. Nilsson, M., Unden, A. B., Krause, D., Malmqwist, U., Raza, K., Zaphiropou-
los, P. G. & Toftgard, R. (2000) Proc. Natl. Acad. Sci. USA 97, 3438-3443.
26. Grachtchouk, M., MO, R., YU, S., Zhang, X., Sasaki, H., Hui, C. C. & Dlugosz,
A. A. (2000) Nat. Genet. 24, 216-217.
27. Gailani, M. R., Stahle-Backdahl, M., Leffell, D. J., Glynn, M., Zaphiropoulos,
P. G., Pressman, C., Unden, A. B., Dean, M., Brash, D. E., Bale, A. E. &
Toftgard, R. (1996) Nat. Genet. 14, 78-81.
28. Oro, A. E., Higgins, K. M., Hu, Z., Bonifas, J. M., Epstein, E. H., Jr., & Scott,
M. P. (1997) Science 276, 817-821.
29. Fan, H., Oro, A. E., Scott, M. P. & Khavari, P. A. (1997) Nat. Med. 3, 788-792.
30. Xie, J., Murone, M., Luoh, S. M., Ryan, A., Gu, Q., Zhang, C., Bonifas, J. M.,
Lam, C. W., Hynes, M., Goddard, A., et al. (1998) Nature 391, 90-92.
31. Chan, E. F., Gat, U., McNiff, J. M. & Fuchs, E. (1999) Nat. Genet. 21, 410-413.
32. Polakis, P. (2000) Genes Dev. 14, 1837-1851.
33. Jiang, J. & Struhl, G. (1998) Nature 391, 493-496.
34. Price, M. A. & Kalderon, D. (2002) Cell 108, 823-835.
35. Andl, T., Reddy, S. T., Gaddapara, T. & Millar, S. E. (2002) De~. Cell 2,
643-653.
36. Mullor, J. L., Dahmane, N., Sun, T. & Ruiz i Altaba, A. (2001) C~c~: Biol. 11,
769-773.
37. Zouboulis, C. C., Seltmann, H., Neitzel, H. & Orfanos, C. E. (1999) J. Invest.
Dermatol. 113, 1011-1020.
38. Zhu, A. J. & Watt, F. M. (1999) Development (Cambridge, U.K) 126, 2285-
2298.
39. Braga, V. M., Hodivala, K. J. & Watt, F. M. (1995) Cell Adhes. Commun. 3,
201-215.
40. Funayama, N., Fagotto, F., McCrea, P. & Gumbiner, B. M. (1995) J. Cell Biol.
128, 959-968.
41. Von Ballestrem, C. G., Uniyal, S., McCormick, J. I., Chau, T., Singh, B. & Chan,
B. M. (1996) Hybridoma 15, 125-132.
42. Toftgard, R. (2000) Cell. Mol. Life Sci. 57,1720-1731.
43. Ghali, L., Wong, S. T., Green, J., Tidman, N. & Quinn, A. G. (1999) J. Invest.
Dermatol. 113, 595-599.
44. Taipale, J., Chen, J. K., Cooper, M. K., Wang, B., Mann, R. K., Milenkovic, L.,
Scott, M. P. & Beachy, P. A. (2000) Nat~cre 406,1005-1009.
11880 1 www.pnas.org/cgi/doi/ 10.1 073/pnas. 1834202100
45. Berman, D. M., Karhadkar, S. S., Hallahan, A. R., Pritchard, J. I., Eberhart,
C. G., Watkins, D. N., Chen, J. K., Cooper, M. K., Taipale, J., Olson, J. M. &
Beachy, P. A. (2002) Science 297, 1559-1561.
46. Chen, J. K., Taipale, J., Cooper, M. K. & Beachy, P. A. (2002) Genes Dev. 16,
2743-2748.
47. Miao, N., Wang, M., Ott, J. A., D'Alessandro, J. S., Woolf, T. M., Bumcrot,
D. A., Mahanthappa, N. K. & Pang, K. (1997) J. Neurosci. 17, 5891-5899.
48. Ahlgren, S. C. & Bronner Fraser, M. (1999) C~c'': Biol. 9, 1304-1314.
49. Cobourne, M. T., HarUcastle, Z. & Sharpe, P. T. (2001) J. De'~t. Res. 80,
1974-1979.
50. Yao, H. H., Whoriskey, W. & Capel, B. (2002) Genes De~. 16, 1433-1440.
51. Lassar, A. B. & Munsterberg, A. E. (1996) Cu~r. Opin. Neurobiol. 6, 57-63.
52. Cossu, G. & Borello, U. (1999) EMBO J. 18, 6867-6872.
53. Kogerman, P., Grimm, T., Kogerman, L., Krause, D., Unden, A. B., Sandstedt,
B., Toftgard, R. & Zaphiropoulos, P. G. (1999) Nat. Cell Biol. 1, 312-319.
54. Kroft, T. L., Patterson, J., Won Yoon, J., Doglio, L., Walterhouse, D. O.,
Iannaccone, P. M. & Goldberg, E. (2001) Biol. Reprod. 65, 1663-1671.
55. Mao, J., Maye, P., Kogerman, P., Tejedor, F. J., Toftgard, R., Xie, W., Wu, G.
& Wu, D. (2002) J. Biol. Chem. 277, 35156-35161.
56. Kanda, S., Mochizuki, Y., Suematsu, T., Miyata, Y., Nomata, K. & Kanetake,
H. (2003) J. Biol. Chem. 278, 8244-8249.
57. Kenney, A. M. & Rowitch, D. H. (2000) Mol. Cell. Biol. 20, 9055-9067.
58. Yoon, J. W., Kita, Y., Frank, D. J., Majewski, R. R., Konicek, B. A., Nobrega,
M. A., Jacob, H., Walterhouse, D. & Iannaccone, P. (2002) J. Biol. Chen~. 277,
5548-5555.
59. Fan, H. & Khavari, P. A. (1999) J. Cell Biol. 147, 71-76.
60. Johnson, P. J. & Heckler, F. (1998) A'~. Plast. S'c~g. 40, 676-677.
61. Duval, A., Gayet, J., Zhou, X. P., Iacopetta, B., Thomas, G. & Hamelin, R.
(1999) Cancer Res. 59, 4213-4215.
62. Takamoto, N., Zhao, B., Tsai, S. Y. & DeMayo, F. J. (2002) Mol. E'Zdocfinol.
16, 2338-2348.
63. Matsumoto, H., Zhao, X., Das, S. K., Hogan, B. L. & Dey, S. K. (2002) De~.
Biol. 245, 280-290.
64. Lewis, M. T., Ross, S., Strickland, P. A., Sugnet, C. W., Jimenez, E., Scott, M. P.
& Daniel, C. W. (1999) Developn7ent (Cambridge, U.K) 126, 5181-5193.
65. Zouboulis, Ch. C. (2000) Ho~m. Res. 54, 230-242.
66. Deplewski, D. & Rosenfield, R. L. (2000) Endocr. Rev. 21, 363-392.
67. Yang, F., Li, X., Sharma, M., Sasaki, C. Y., Longo, D. L., Lim, B. & Sun, Z.
(2002) J. Biol. Chem. 277, 11336-11344.
68. Pawlowski, J. E., Ertel, J. R., Allen, M. P., Xu, M., Butler, C., Wilson, E. M.
& Wierman, M. E. (2002) J. Biol. Chem. 277, 20702-20710.
69. Chesire, D. R., Ewing, C. M., Gage, W. R. & Isaacs, W. B. (2002) Oncogene
21, 2679-2694.
70. Braun, K. M., Niemann, C., Jensen, U. B., Sundberg, J. P., Silva-Vargas, V. &
Watt, F. M. (2003) Developme~nt (Cambridge, U.K), in press.
Niemann et a/.
Representative terms from entire chapter:
hair follicle
