Differential regulation of epidermal function by VDR coactivators

Abstract

The transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator and corepressor complexes, which bind to the VDR in a ligand (1,25(OH)2D3) dependent (coactivators) or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the vitamin D interacting protein (DRIP) complex and the steroid receptor coactivator (SRC) complexes. These coactivator complexes are not interchangeable in their regulation of keratinocyte proliferation and differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating keratinocyte, whereas SRC2 and 3 and their associated proteins are the major coactivators binding to VDR in the differentiated keratinocyte. Moreover, we have found a specific role for DRIP205 in the regulation of β-catenin pathways regulating keratinocyte proliferation, whereas SRC3 uniquely regulates the ability of 1,25(OH)2D3 to induce more differentiated functions such as lipid synthesis and processing required for permeability barrier formation and the innate immune response triggered by disruption of the barrier. These findings provide a basis by which we can understand how one receptor (VDR) and one ligand (1,25(OH)2D3) can regulate a large number of genes in a sequential and differentiation specific fashion.

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Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 308–313
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Differential regulation of epidermal function by VDR coactivators?
D.D. Biklea,b,∗, A. Teicherta, L.A. Arnoldc, Y. Uchidab, P.M. Eliasb, Y. Odaa
aEndocrine Research Unit, Department of Medicine, Veterans Affairs Medical Center, San Francisco, CA 94121, USA
bDepartment of Dermatology, Veterans Affairs Medical Center, San Francisco, CA 94121, USA
cDepartment of Chemical Biology and Therapeutics, St. Jude Children’s Hospital, Memphis, TN 38105, USA
a r t i c l ei n f o
Article history:
Received 13 November 2009
Received in revised form 6 March 2010
Accepted 8 March 2010
Keywords:
Vitamin D receptor
Coactivators
Keratinocytes
Differentiation
Proliferation
?-Catenin
Innate immunity
Permeability barrier
a b s t r a c t
The transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator
and corepressor complexes, which bind to the VDR in a ligand (1,25(OH)2D3) dependent (coactivators)
or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the
vitamin D interacting protein (DRIP) complex and the steroid receptor coactivator (SRC) complexes.
Thesecoactivatorcomplexesarenotinterchangeableintheirregulationofkeratinocyteproliferationand
differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating
keratinocyte,whereasSRC2and3andtheirassociatedproteinsarethemajorcoactivatorsbindingtoVDR
inthedifferentiatedkeratinocyte.Moreover,wehavefoundaspecificroleforDRIP205intheregulationof
?-catenin pathways regulating keratinocyte proliferation, whereas SRC3 uniquely regulates the ability of
1,25(OH)2D3to induce more differentiated functions such as lipid synthesis and processing required for
permeability barrier formation and the innate immune response triggered by disruption of the barrier.
These findings provide a basis by which we can understand how one receptor (VDR) and one ligand
(1,25(OH)2D3) can regulate a large number of genes in a sequential and differentiation specific fashion.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
The epidermis is composed of four layers of keratinocytes at
different stages of differentiation (Fig. 1). The basal layer (stra-
tum basale, SB) rests on the basal lamina separating the dermis
and epidermis. Within this layer are the stem cells. These cells
proliferate, providing the cells for the upper differentiating layers.
They contain an extensive keratin network comprised of keratins
K5 and K14. The layer above the basal cells is the spinous layer
(stratum spinosum, SS). These cells initiate the production of the
keratins K1 and K10, which are the keratins characteristic of the
moredifferentiatedlayersoftheepidermis.Cornifiedenvelopepre-
cursors such as involucrin also appear in the spinous layer as does
the enzyme transglutaminase, responsible for the ?-(?-glutamyl)
lysine cross-linking of these substrates into the insoluble cornified
envelope.Thegranularlayer(stratumgranulosum,SG),lyingabove
the spinous layer, is characterized by electron-dense keratohyalin
granules containing profilaggrin, the precursor of filaggrin, a pro-
?Special issue selected article from the 14th Vitamin D Workshop held at Brugge,
Belgium on October 4–8, 2009.
∗Corresponding author at: Endocrine Research Unit, Department of Medicine,
Veterans Affairs Medical Center, 4150 Clement St. (111N), San Francisco, CA 94121,
USA.
E-mail address: daniel.bikle@ucsf.edu (D.D. Bikle).
tein thought to facilitate the aggregation of keratin filaments, and
loricrin,amajorcomponentofthecornifiedenvelope.Thegranular
layer also contains lamellar bodies, lipid-filled structures that fuse
withtheplasmamembrane,divestingtheircontentsintotheextra-
cellularspacebetweentheSGandstratumcorneum(SC)wherethe
lipid contributes to the permeability barrier of skin. The outer layer
oftheepidermisprovidesbothabarriertowaterloss(permeability
barrier) and a barrier to invasion by infectious organisms (innate
immunity).
The keratinocytes not only produce vitamin D3 from 7-
dehydrocholesterol (7-DHC) but convert D3to its active metabolite
1,25(OH)2D3. These cells also possess the receptor for 1,25(OH)2D3
(VDR), enabling them to respond to 1,25(OH)2D3in an intracrine,
autocrine or paracrine fashion [1]. 1,25(OH)2D3increases involu-
crin, transglutaminase activity, loricrin, filaggrin and cornified
envelope formation at subnanomolar concentrations [2–7], while
inhibiting proliferation at least at concentrations above 1nM. In
addition, 1,25(OH)2D3induces the enzymes responsible for pro-
cessing the long chain glucosylceramides that are critical for
permeability barrier formation [8] and the toll-like receptors
(TLR2), coreceptor CD14, and defensins critical for the innate
immune response in skin [9].
Theprocessofepidermal
1,25(OH)2D3 and VDR regulate all steps from the control of
proliferation in the SB, to the regulation of K1, K10, involucrin,
and transglutaminase production in the SS, to the regulation of
differentiationissequential.
0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
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Fig.1. Amodelshowingthedifferentialexpressionofvariousmarkersandfunctions
in the epidermis with differentiation. DRIP205 is most highly expressed in the stra-
tumbasaleandspinosumwhereitparticipateswithVDRinregulatingproliferation.
SRC3ontheotherhandisfoundinhighestconcentrationinthestratumgranulosum
whereitparticipateswithVDRintheregulationofterminaldifferentiationincluding
formation of the permeability barrier and innate immune responsiveness.
loricrin and filaggrin production in the SG, to the synthesis of
lipids required for the permeability barrier in the SC, and to the
development of the innate immune system [1,7,9]. The major
question being addressed in our current studies is how does this
occur? In previous studies we noted the differential distribution
of VDR coactivators DRIP 205, SRC2 and 3 within the epidermis,
and their differential binding to VDR depending on the state
of differentiation of the keratinocyte [10–12]. In this study we
show that in addition to differential distribution, these coactivator
complexes are differentially selective for genes regulated by
VDR. Our data support the hypothesis that SRC facilitates the
ability of VDR to regulate the more differentiated functions of the
keratinocyte including barrier formation and the innate immune
response, whereas DRIP facilitates the ability of VDR to regulate
proliferation.
2. Materials and methods
2.1. Keratinocyte cultures
Normal epidermal keratinocytes were isolated from neonatal
human foreskin, and grown in 154CF medium (KGM, Cascade).
The cells were cultured with KGM containing 0.03mM calcium
to obtain proliferating keratinocytes; differentiation was induced
with 1.2mM calcium, and in some experiments with ascorbic acid
and serum to maximize lamellar body formation [13].
2.2. VDR and coactivator knockdown and overexpression
FourindividualsiOligos(Dharmacon)wereinitiallytestedalone
and in combination for VDR and the coactivators (except for SRC3
which used a previously published sequence), and the best combi-
nation was chosen for efficient knockdown of gene expression. For
long-term knockdown studies, the most efficient oligonucleotide
was subcloned into an adenovirus [7]. Constitutive expression of
VDR was achieved by cloning a translational fusion of VDR to
green fluorescent protein (GFP) into pDNR-CMV (BD Biosciences)
for subsequent cloning into the adenoviral host [7]. Primary sec-
ond passage human keratinocytes were transfected at a rate of
75–250ifu/cell and grown for 3 days in 0.03M Ca2+KGM. Depend-
ing on the experiment they were then transferred into media
containing 1.2mM Ca2+and/or treated with vehicle or 10−8M
1,25(OH)2D3.
2.3. Morphologic assessment
Routine phase microscopy was used to visualize cell morphol-
ogy. Confocal microscopy was used to colocalize ?-catenin and
E-cadherin in cellular membranes. Ultrastructural examination
was performed to look for changes in cell–cell adhesion, lamel-
lar body (LB) secretion, and barrier disruption. LB density and its
content, lipid secretion and its location, and lipid processing were
assessed visually in randomly photographed micrographs.
2.4. Cell proliferation and apoptosis
Proliferation was assessed by BrdU incorporation using the
Zymed BrdU staining kit according to the manufacturer’s instruc-
tions. The identification of apoptotic cells was determined using
TACSTM2 TdT-DAB In Situ Apoptosis Detection kit from Trevigen.
2.5. mRNA and protein levels
ThemRNAlevelsofvariousgenescriticalforkeratinocyteprolif-
erationanddifferentiationweremeasuredbyquantitativerealtime
PCRusingstandardproceduresaswehavepreviouslydescribed[7].
The protein levels were analyzed by Western blots using standard
techniques available in our laboratory [14].
2.6. Lipid analysis
Epidermal specific lipids were measured in the epidermis and
cultured keratinocytes using high performance thin-layer chro-
matography as we have recently published [8].
2.7. Innate immune response
Skin wounds were generated by a 5mm full thickness inci-
sion under aseptic conditions. A small biopsy was subsequently
obtained and processed by qRTPCR, Western blot analysis and
immunohistochemistry for cathelicidin, CD14, and toll-like recep-
tors using methods described above.
3. Results
3.1. The role of VDR and its coactivators in regulating
proliferation and apoptosis
The function of DRIP205 and SRC3 in regulating keratinocyte
proliferation and apoptosis was examined using siRNA-silencing
technology.Humanepidermalkeratinocytesweretransfectedwith
siRNA for VDR, DRIP205, or SRC3 to block their expression. Effi-
ciencyofknockdownwasatleast75%(Fig.2AandB).BothDRIP205
and VDR silencing resulted in changes in keratinocyte morphology
(fromcuboidaltospindle-shapedcells),butknockdownofSRC3did
not (Fig. 2C, phase contrast). Keratinocyte proliferation was then
assessed by BrdU incorporation demonstrating that knockdown of
VDR and DRIP205 increased proliferation, whereas knockdown of
SRC3 had the opposite effect (Fig. 2C and D). Apoptosis as assessed
byTUNELstaining,ontheotherhand,wasdecreasedbyknockdown
of VDR and DRIP, whereas SRC3 knockdown increased apoptosis
(Fig. 2E).
?-Catenin, by inducing genes such as cyclin D1 and Gli1 (an
importanttranscriptionfactorinthehedgehogpathway),promotes
theproliferationofkeratinocytes.Thesegenesareknowntocontain
bothLEF/TCFandVDRresponseelements[15].Therefore,toexplore
the mechanism by which silencing of DRIP205 and VDR results in
hyperproliferation, we investigated their roles in ?-catenin sig-
naling. Initial experiments demonstrated that 1,25(OH)2D3and
VDR overexpression inhibited the expression of ?-catenin reporter
constructs (cyclin D1 promoter, TOPGlow) (data not shown). Fol-
lowing these studies we examined whether DRIP205 played a role
in the regulation of cyclin D1 and Gli1 in keratinocytes. These
results are shown in Fig. 3. The data demonstrated that silencing
of DRIP205 increased the expression of both cyclin D1 and Gli1,

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Fig. 2. Impact of VDR, DRIP205, and SRC3 knockdown on keratinocyte morphology, proliferation and apoptosis. The keratinocytes were transfected with siRNAs to VDR,
DRIP205, and SRC3, respectively. The efficiency and specificity of the knockdowns are shown at both the mRNA (A) and protein (B) level. Phase contrast was used to assess
morphology(C).ProliferationwasevaluatedusingBrdU(CandD),andapoptosiswasdeterminedbyTUNELstaining(E).Statisticalsignificanceisshownbyasterisks(*P<0.05;
**P<0.01).
like that seen with the silencing of VDR. SRC3 knockdown did
not show this response (data not shown). Although 1,25(OH)2D3
tended to suppress cyclin D1 and Gli1 expression in the cells in
which VDR and DRIP205 had been silenced, presumably because
the knockdown was incomplete, these effects of 1,25(OH)2D3were
nolongersignificant.Usingconfocalmicroscopy,wethenobserved
thatknockdownofVDRandDRIP205butnotSRC3blockedcalcium
induced formation of the E-cadherin/?-catenin complex in the
membrane, suggesting that by keeping ?-catenin anchored to the
membrane,lesswasavailabletostimulateproliferationthroughits
Fig. 3. Regulation by VDR and DRIP205 of cyclin D1 and Gli1 expression. The ker-
atinocytes were transfected with the specific and control siRNAs to VDR, DRIP205,
and SRC3. The cells were treated with 10nM 1,25(OH)2D3or vehicle as in Fig. 2.
The mRNA levels of cyclin D1, Gli1, and control L19 were measured by real time
PCR. The expression is shown as % of the sicontrol vehicle cultures. The error bars
enclosemean±SDoftriplicatecultures.Statisticalsignificanceisshownbyasterisks
(*P<0.05; **P<0.01).
nuclear interactions with LEF/TCF (data not shown). These results
indicated that DRIP205 played a greater role than SRC3 in medi-
ating VDR regulation of keratinocyte proliferation at least in part
through regulation of ?-catenin signaling.
3.2. Effect of VDR and coactivators on differentiation markers
To evaluate the role of these coactivators in keratinocyte differ-
entiation keratinocytes were maintained in high calcium for up to
7 days. Differentiation was evaluated by measuring mRNA levels
of various differentiation markers. These results are found in Fig. 4.
After 3 days of culture, levels of the early markers keratin 1 (K1)
and keratin 10 (K10) increased. When DRIP205, SRC3 or VDR was
silenced, the induction of K1 and K10 was significantly inhibited.
Latedifferentiationmarkers,filaggrin(FLG)andloricrin(LOR)were
inducedafter7daysofculture.Asfortheearlydifferentiationmark-
ers, silencing of DRIP205, SRC3 or VDR markedly inhibited their
induction by calcium. These results indicated that both DRIP205
and SRC3 play critical roles in the induction of these markers of
keratinocyte differentiation.
3.3. Effect of VDR on permeability barrier formation
One of the most differentiated functions of the keratinocyte is
the formation of the permeability barrier. This involves the for-
mation of the cornified envelope, a process in which proteins
such as involucrin and loricrin are crosslinked into an insoluble
matrix, and the production and secretion of long chain lipids, glu-
cosylceramides in particular, into this matrix to water proof it. To
examine whether DRIP and SRC complexes differ in their roles in
the production and secretion of the lipids for barrier formation, a
vitaminCandserumsupplementedsubmergedculturesystemwas
employed,inwhichkeratinocytesformbarrierlayerswithlamellar
bodies resembling those of the epidermis in vivo. The expression
of DRIP205, SRC3, and VDR was blocked using adenovirus encoding

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Fig. 4. Impact of DRIP205, SRC3, and VDR silencing on calcium induced expression of keratinocyte differentiation markers. The keratinocytes were transfected with siRNAs
to VDR, DRIP205, and SRC3, respectively. The cells were then switched to 1.2mM calcium, and the mRNA levels of the indicated differentiation markers determined at 3
and 7 days by qRTPCR. Silencing of both DRIP205 and SRC3, as well as VDR inhibited the calcium induced expression of these markers. The error bars enclose mean±SD of
triplicate cultures, and in most cases are within the symbol.
shRNA for each coactivator (75ifu/cell) or the control adenovirus.
The blocking efficiency for VDR and the coactivators was VDR 70%,
DRIP 90%, SRC3 80% as assessed by protein levels [7]. The infected
keratinocytes were maintained in vitamin C supplemented serum
for an additional 5–10 days to allow formation of the multilayered,
epidermis-like tissue. The results are shown in Fig. 5.
Wefirstassessedtherelativelevelsoftheimmediateprecursors
of the barrier ceramides, i.e. the glucosylated ceramides (GlcCer)
(Fig. 5A). When VDR was silenced, total GlcCer and epidermis
specific GlcCer (acylGlcCer) levels decreased. Silencing SRC3, but
not DRIP also decreased total GlcCer and epidermis specific acyl-
Cer species. We then used electron microscopy to examine these
cells for their ability to carry out three key steps in barrier forma-
tion: (i) stratification, (ii) lamellar body (LB) formation, (iii) lipid
secretion processing. The results are shown in Fig. 5B. Stratifica-
tion was assessed as the extent of formation of organelle depleted
Fig. 5. VDR and SRC3 but not DRIP205 regulate production of epidermal specific GlcCer species, the formation of lamellar bodies, and the key enzymes and transporter
involved. Human epidermal keratinocytes were infected with shRNA adenovirus for VDR and its coactivators to block their expression. Keratinocytes were maintained in
medium supplemented with vitamin C and serum to induce LB production. (A) The levels of key lipids were determined expressed as % of control. Data are represented as
mean±SD of three measurements. (*P<0.03). (B) The tissues were examined by electron microscopy to determine number of LBs and their content (left panels). The lipid
secretion and processing were evaluated using ruthenium staining (right panels). Bars represent 1.0?m (left panels) and 0.2?m (right panels). The table summarizes the
results. (C) The transcription of lipid synthesis enzymes was evaluated by qRTPCR. The mRNA levels were normalized to control gene L19, and relative expression to control
group was expressed as a percentage (%). Only the significantly affected genes are shown (ELOVL4, UGCG, and ABCA12). Data are represented as mean±SD of at least three
independent experiments (*P<0.01).

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Fig. 6. The innate immune response in the epidermis requires 1,25(OH)2D3and the coactivator SRC3. (A) The expression of cathelicidin, CD14, CYP27b1, and TLR2 (but not
other TLRs) in human skin was increased by wounding. (B) This experiment was repeated in CYP27b1 null mice demonstrating that CYP27b1 was required for the induction
of CD14 and cathelicidin. (C) Cultured human keratinocytes were used to demonstrate the ability of 1,25(OH)2D3(100nM) to stimulate TLR2, CD14, and cathelicidin. (D)
Silencing of SRC3 but not of DRIP205 blocked the ability of 1,25(OH)2D3to induce cathelicidin expression in these cultured human keratinocytes. All data are expressed as
mean±SD of triplicate experiments.
flattened corneocyte layers enclosed by a cornified envelope. LB
formation was evaluated as the number, density (per ?m2cytosol)
and lamellar contents of individual LBs in osmium tetroxide post-
fixed samples. LB secretion and processing of secreted lipids into
lamellar bilayers within the extracellular spaces were assessed by
post-fixation with ruthenium tetroxide. Control tissues infected
withcontroladenovirusshowedsignificantstratificationandcorni-
fication, as well as a reasonable density of LBs, some of which
secreted their contents into the extracellular spaces (Fig. 5B, sicon-
trol). Most of these organelles exhibited internal lamellar contents.
In addition, organelle secretion and post-secretory processing into
mature extracellular lamellar membrane structures were readily
observed. In contrast, when VDR is silenced, both incomplete strat-
ification and abnormal lamellar bilayer formation were observed
(Fig. 5B, siVDR). In these VDR silenced tissues, we also found
decreased numbers of LBs, and individual LBs often lacked typi-
cal lamellar contents. In addition, very little LB secretion appeared
to occur into the extracellular spaces in VDR silenced cells. SRC3
silenced cells showed more marked abnormalities, with no strati-
fication, no LB formation, and no lamellar bilayers (Fig. 5B, siSRC3).
When DRIP205 was blocked, stratification was incomplete, but a
reasonable number of LBs, replete with internal lamellar content
and relatively normal appearing lamellar bilayers, were observed
(Fig. 5B, siDRIP). The results are summarized in the table contained
in Fig. 5. To examine the mechanism by which VDR and SRC3 regu-
late glucosylceramide synthesis and barrier formation in cultured
keratinocytes we measured changes in the expression of the key
enzymes involved (Fig. 5C). We found that the mRNA level of the
fatty acid elongase ELOVL4, essential for N-acyl fatty acid chain
elongation and a key step in acylCer and acylGlcCer synthesis,
was significantly decreased when either VDR or SRC3 was silenced
but not when DRIP205 was silenced (Fig. 5C). Furthermore, the
expression and enzyme activity of ceramide glucosyl transferase
(UGCG) and the lipid transporter, ABCA12, responsible for trans-
porting lipids into LB, were selectively reduced by silencing VDR
and SRC3, but not DRIP205 (Fig. 5C). Together, these results indi-
cate that VDR and SRC3 but not DRIP205 regulate specific enzymes
in the acylCer/GlcCer synthetic pathway, including steps involved
in the elongation of fatty acids (ELOVL4), Cer glucosylation (UGCG)
and lipid loading into LB (ABCA12).
3.4. Role of 1,25(OH)2D3/VDR and its coactivators in the innate
immune response of the epidermis
The barrier function of the epidermis includes its resistance
to pathogenic organisms. Part of this defense involves the innate
immune system. Innate immune responses involve the activation
of toll-like receptors (TLRs) that interact with specific membrane
patterns (PAMP) shed by infectious agents that trigger the innate
immune response in the host. Activation of TLRs leads to the induc-
tion of antimicrobial peptides such as cathelicidin and reactive
oxygen species, which kill the organism (Fig. 6 model). In pre-
vious studies we [9,12] have shown that 1,25(OH)2D3is critical
for this system to work. In these studies skin was wounded with
a small incision. This induced the expression of TLR2 and CD14
(key receptors in the innate immune response), cathelicidin, and
CYP27b1(theenzymeproducing1,25(OH)2D3)intheskin(Fig.6A).
The induction of CD14 and cathelicidin by wounding the skin of
mice lacking CYP27b1 did not occur (CD14) or was blunted (cathe-
licidin) (Fig. 6B). We then showed in human keratinocytes that

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1,25(OH)2D3induced TLR2, CD14, and cathelicidin (Fig. 6C and D),
and finally, that this action of 1,25(OH)2D3/VDR was blocked in
keratinocytes in which SRC3 was silenced, although not in ker-
atinocyteslackingDRIP205(Fig.6D).Theseresultssuggestthatlike
formation of the lipids required for the permeability barrier, the
innate immune response requires SRC3, not DRIP205.
4. Discussion
In this study we explored the different roles of the coactivators
DRIP205andSRC3inmodulating1,25(OH)2D3/VDRtranscriptional
activity for a number of genes involved with proliferation and
differentiation of keratinocytes. This approach was motivated by
our initial findings that DRIP binding to VDR dominated extracts
from proliferating keratinocytes, whereas SRC3 binding to VDR
dominated extracts from differentiated keratinocytes. We then
proceeded to demonstrate that DRIP205 was mainly expressed in
proliferating keratinocytes, whereas SRC3 was mainly expressed
in differentiated keratinocytes both in vitro and in vivo. This dif-
ferential distribution has functional consequences. Although some
genes or keratinocyte functions such as CYP24 expression and
a number of differentiation markers appeared to be regulated
equally well whether DRIP205 or SRC3 was the coactivator, other
genes/functions clearly showed preferences. For example, prolifer-
ation of keratinocytes and the regulation of genes associated with
proliferation such as cyclin D1 and Gli1 clearly favored DRIP205,
in that knockdown of SRC3 unlike that of DRIP205 failed to have
a significant effect on proliferation. On the other hand, more dif-
ferentiated functions such as formation of the permeability barrier
and development of the innate immune response favored SRC3,
in that knockdown of DRIP205 unlike that of SRC3 failed to alter
these processes or the induction by 1,25(OH)2D3/VDR of the genes
involved in these processes. These results support our hypothesis
that DRIP205 and SRC3 differentially regulate 1,25(OH)2D3/VDR
actions in keratinocytes such that the appropriate actions are
accomplished in the appropriate cell—i.e. proliferation is regulated
inproliferatingkeratinocytesusingDRIP205ascoactivatorwhereas
terminal differentiation and maintenance of the protective barrier
is regulated in differentiated keratinocytes using SRC3 as coactiva-
tor.
At this point these studies have involved keratinocytes in vitro.
We have developed mice selectively null for DRIP205 or SRC3 in
theirkeratinocytes.DRIPnullmiceshowhyperproliferationofboth
hair follicles and epidermis similar to VDR null mice during first 3
weeks of life, but with time develop alopecia as the hair follicles
subsequently atrophy again like VDR null mice. SRC3 null mice do
notshowsuchchanges.OntheotherhandSRC3nullmicehaveboth
a defective permeability barrier due to changes in lipid processing
in the epidermis, and fail to mount an innate immune response
to wounding. Thus the in vitro experiments reported here have
successfully predicted the changes observed in vivo.
Acknowledgements
The authors acknowledge the financial support of NIH
PO1AR39448,RO1AR050023,VAMeritReview,andAmericanInsti-
tute of Cancer Research 07A140 and the administrative support
from Ms. Teresa Tong and Victoria Lee.
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