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Departments of Molecular and Cellular Biology [K. M. L., X-J. W., D. R. R.] and Dermatology [X-J. W., D. R. R.] and Program in Developmental Biology [M. I. K., D. R. R], Baylor College of Medicine, Houston, Texas 77030, and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 [A. Y., F. M.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionNote Added in ProofREFERENCES |
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Np63
, decreased
dramatically in normal keratinocytes or newborn epidermis at both the
protein and RNA levels after UV-B irradiation. In an attempt to further
investigate the significance of the UV-B-induced decrease of this p63
isoform as well as further delineate the function of p63 in the
epidermis, we generated transgenic mice that constitutively express
Np63 in the mouse epidermis using the loricrin promoter
(ML.Np63). The ML.Np63 mouse epidermis developed normally,
with no overt phenotype and an unaltered proliferation rate. When
challenged by UV-B exposure, the ML.Np63 mice exhibited a
40–45% decrease in the number of apoptotic cells in the epidermis as
compared with nontransgenic littermates. These results suggest that
aberrant expression of Np63 altered the UV-B-induced apoptotic
pathway in the transgenic epidermis, proving that down-regulation of
Np63 in response to UV-B is important to epidermal apoptosis. The
forced overexpression of Np63 may act via a dominant negative
effect on the endogenous p53 transcriptional activity required for
UV-B-induced apoptosis. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofREFERENCES |
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50% of all human cancers (3)
, demonstrating the
universality of this tumor suppressor. DNA damage can be caused by a number of genotoxic agents, and UV-B radiation is one of the most biologically relevant inducers of DNA damage. UV-B radiation in sunlight is the carcinogen responsible for most human skin cancers (4 , 5) . In response to the DNA damage induced by UV-B, the p53 protein is stabilized and translocated to the nucleus, where it triggers an arrest of the cell cycle or induces apoptosis (1 , 4 , 6) . In the epidermis, the response to UV-B irradiation is frequently characterized by the induction of apoptosis, primarily mediated by p53 (7) . In the absence of p53, fewer apoptotic cells are observed in the epidermis in response to UV-B (7) . Also, mice null for p53 are more susceptible to UV-B-induced tumorigenesis than wild-type mice, implying a critical function for p53 in combating the detrimental effects on the epidermis of this carcinogen (8 , 9) .
Recently, two homologues of p53, p63 and p73, have been discovered and
cloned, generating a new family of p53-like genes.
The p63 gene shares extensive homology to p53 and produces
multiple transcripts with varying functions (10, 11, 12, 13)
.
Unlike the fairly ubiquitous nature of the p53 protein, p63 exhibits a
rather tissue-specific distribution in that it is most detectable in
the basal layer of stratified epithelia, including the epidermis
(13)
. It is reported that the most highly expressed p63
isoform in the epidermis is Np63 (13)
. This isoform
lacks the 5' region that exhibits extensive homology to the
transactivation domain of p53. In contrast to p53, Np63 fails to
induce apoptosis when overexpressed in cultured cells. Np63 has
also been shown to inhibit p53 transcriptional activity
(13)
. The p63 gene was disrupted by homologous
recombination, resulting in severe limb, craniofacial, and epithelial
defects and leading to death shortly after birth (14
, 15)
.
The newborns lacking functional p63 did not have a recognizable
epidermis or hair follicles but possessed a thin, single cell layer
covering the body (14
, 15) . This cell layer did not
exhibit epidermal characteristics such as keratin expression, but in
one model, this cell layer showed a few isolated patches of highly
differentiated clusters of epidermal-like cells (14
, 15)
.
Because p63 is essential to normal epidermal development and is a
homologue of the p53 tumor suppressor, we investigated the role of p63
in the epidermis in response to UV-B irradiation. We found that in
contrast to p53, Np63 is down-regulated in response to UV-B. To
further characterize the importance of this down-regulation as well as
the functional importance of p63 in the epidermis, we generated
transgenic mice that overexpress Np63 in the epidermis. The
epidermis of these transgenic mice developed normally but exhibited a
reduction in UV-B-induced apoptosis, indicating that the
down-regulation of this p63 isoform is critical for normal epidermal
UV-B-induced apoptosis.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofREFERENCES |
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UV-B Treatment of Keratinocytes and Mice.
Before exposure to UV radiation, the media were removed, and cells were
rinsed twice with Dulbecco’s PBS (Life Technologies, Inc.,
Gaithersburg, MD) and covered with 1.5 ml of PBS during the UV
treatment. Cells were exposed to 50 mJ/cm2
UV-B
from FS40T12 bulbs (National Biological/ETA Systems, Twinsburg, OH) as
monitored by a radiometer/photometer (International Light, Inc,
Newburyport, MA). PBS was removed, and media were added to cells as
soon as UV treatment was complete. Samples were taken at 0 (no UV
radiation), 6, 12, and 24 h after UV treatment. Culture dishes
were rinsed three times with PBS, and PBS was removed before freezing
the dish on dry ice and storing it at -80°C until protein or RNA
extraction was performed. Newborn pups were immobilized, and back skin
was exposed to 100 mJ/cm2
UV-B from FS40T12 lamps
(National Biological/ETA Systems) as monitored by a
photometer/radiometer (International Light, Inc.). Samples of back skin
were taken at 0 (no UV radiation), 6, 12, and 24 h after UV
exposure for normal ICR pups or at 24 h after UV exposure
for transgenic litters. Portions of the skin were frozen in OCT, fixed
in 10% neutral buffered formalin, and embedded in paraffin or frozen
in liquid nitrogen for storage. Adult mice were exposed to 100
mJ/cm2
UV-B, monitored at the height of the ears.
Ear samples were processed as described here for newborn back skin.
Protein Extraction and Western Blotting.
Protein was extracted from cultured keratinocytes and neonatal mouse
epidermis and subjected to Western blotting as described previously
(17)
. Membranes were blocked with TBST/NFDM [50
mM Tris (pH 7.5), 150 mM NaCl, 0.075% Tween 20
(Sigma, St. Louis, MO), and 5% nonfat dried milk] for 30
min at room temperature. Membranes were probed for p53 (Ab-7, sheep
anti-p53; Oncogene Research Products, Cambridge, MA), stripped using
ImmunoPure IgG Elution Buffer (Pierce Chemical Company, Rockford, IL)
according to the manufacturer’s instructions, probed for p63 (mAb4A4,
mouse monoclonal anti-p63; McKeon Laboratory), stripped, and probed for
K142
[sheep anti-K14 (18)
] as a loading control. Ab-7 and
mAb4A4 were diluted 1:2000 or 1:100, respectively, in 1:3 TSBT/NFDM:TBS
and incubated overnight at 4°C. Probing for K14 was performed at room
temperature for 1 h using a 1:500 dilution in 1% NFDM in TBS.
Blots were rinsed with TBS and incubated for 1 h at room
temperature with peroxidase-conjugated secondary antibodies (Sigma)
diluted in 1:3 TBST/NFDM:TBS at 1:2,500 (against p53 and p63) or
1:20,000 (against K14). After rinsing with TBS, blots were exposed to
PicoWest SuperSignal ECL Substrate (Pierce Chemical Company) and
exposed to Biomax MR film (Kodak, Rochester, NY). A quantitative
comparison of protein expression was determined by densitometric
scanning of the films and normalizing each sample to the K14 signal.
RNA Extraction and RPAs.
RNA was extracted from cultured keratinocytes and neonatal mouse
epidermis using RNazol B (TelTest, Friendswood, TX) according to the
manufacturer’s instructions. A 544-bp fragment from the 3'-most end of
p63 (
Fig. 2
) was cloned by RT-PCR from an epidermal RNA sample and
ligated to pGemT-Easy (Promega, Madison, WI) and sequenced. This clone
was linearized with SpeI and transcribed in the presence of
[32P]CTP with T7 RNA polymerase. RPAs were
performed using the RPA II Kit (Ambion, Austin, TX). A
32P-labeled cyclophilin riboprobe was included in
each reaction as a loading control. The intensity of protected product
was determined by densitometric scanning of the films, and samples were
normalized to the cyclophilin signal.
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Np63 Mice.Np63 was modified by PCR to
incorporate amenable restriction sites for cloning into the ML promoter
to generate the ML.Np63 transgene (Fig. 2
). The sequence was
confirmed, the transgene was liberated from plasmid backbone by
restriction digestion with BamHI and gel-purified, and
transgenic mice were generated by standard techniques. Founder mice
were identified by PCR of tail tip DNA using primers ML-1f
(5'-GCTCTTCTGCTCTTCTTCC-3') and 63-2r (5'-GCATCGTTTCACAACCTCG-3'; Fig. 2
).
Detection of ML.Np63 Transgene Expression.
Reverse transcription was performed on 2 µg of epidermal RNA using
random hexamers (Boehringer Mannheim, Indianapolis, IN) and Moloney
murine leukemia virus reverse transcriptase (Promega) according
to the manufacturer’s instructions. PCR reactions were performed using
primers 63-5f (5'-GTGCCTCTACCGTCAGTGTG-3') and mceX46
(5'-ACAACAGAGCTGGAAGAG-3') for 35 cycles of 30 s at 95°C, 1 min
at 60°C, and 1 min at 72°C. RT-PCR results were verified by
immunohistochemistry on newborn epidermis using mAb4A4. Formalin-fixed,
paraffin-embedded sections were deparaffinized in HemoDe:Xylene (3:1),
rehydrated, and treated with HistoMouse Kit reagents 1A (30 min) and 1B
(10 min; Zymed Laboratories, San Francisco, CA). Slides were incubated
with mAb4A4 (1:250 dilution) for 2 h at room temperature followed
by biotinylated horse antimouse (Vector Laboratories, Burlingame, CA)
and ABC Elite reagent (Vector Laboratories). Slides were incubated with
SigmaFast 3,3'-diaminobenzidine (Sigma) and counterstained with
hematoxylin (Gill’s #3; Fisher Biotech, Fair Lawn, NJ). Coverslips
were mounted with GVA mount (Zymed Laboratories).
In Vivo BrdUrd Incorporation and Analysis.
Newborn mice were injected i.p. with 250 µg of BrdUrd (Sigma) in
0.9% sterile saline solution. One h later, mice were sacrificed, and
skin samples were fixed in Carnoy’s fixative or frozen in OCT (Sakuro
Finetek, Torrance, CA). BrdUrd staining was performed as described
previously (19)
.
TUNEL Analysis.
Formalin-fixed, paraffin-embedded samples were sectioned to 8 µm. The
sections were deparaffinized and rehydrated, and TUNEL analysis was
performed using the Apoptosis Detection System, Fluorescein (Promega)
according to the manufacturer’s instructions. Sections were then
stained with propidium iodide (Sigma). The number of apoptotic cells
per millimeter of epidermis was examined.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofREFERENCES |
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Np63, increased slightly over basal
levels (Fig. 1a
). This is the p63 isoform that is the most highly expressed
isoform in the normal epidermis. However, a dramatic decrease at the
protein level of Np63 occurred at 12 h after UV-B treatment (Fig. 1a
). Another p63 protein product, Np63
, was expressed
at low levels before UV-B treatment and was undetectable at 12 and
24 h after UV-B treatment (data not shown). To determine whether
the decrease in p63 protein was a posttranscriptional event or whether
it reflected a decreased level of transcription, RPA was performed. The
p63 transcript was readily detected before UV-B treatment and
decreased sharply after UV-B exposure (Fig. 1b
). This is in
contrast to p53 transcripts, which are detected at a constant level
before and after UV-B exposure (6)
. Similar results were
obtained after exposing normal newborn pups to 100
mJ/cm2
UV-B and analyzing epidermal protein and
RNA (data not shown).
Expression of the ML.Np63 Transgene in the Epidermis.
Because the p63-null mouse does not survive, we were interested in
developing a mouse model that would allow us to further investigate the
significance of this decrease in p63 in response to UV-B and to better
understand the function of p63 in the epidermis. The Np63 cDNA
was placed under the control of the ML promoter (ML.Np63), and
transgenic mice were generated (Fig. 2a
). This transgenic promoter construct was generated in our
laboratory and has been shown to successfully drive expression to the
epidermis in the superbasal layers as well as a portion of the basal
cells (20)
. Three founder lines were identified by PCR of
tail tip DNA: (a) D1588; (b) D1593; and
(c) D1595. To determine whether the transgene was being
expressed, RT-PCR was performed. These three lines were all positive
for the transgene transcript (Fig. 2b
). To confirm
transgenic protein expression, immunohistochemistry was performed on
newborn transgenic and nontransgenic epidermis using mAb4A4. Because
this ML promoter expresses transgenes throughout the epidermis, and
endogenous p63 protein is detected strictly in the basal layer, any p63
detected suprabasally is a result of transgene expression. The
immunohistochemistry confirmed the RT-PCR results, and it appeared that
line D1588 had the highest level of ML.Np63 transgene expression
(Fig. 2c
). In this line, most suprabasal cells showed a
strong p63 signal, whereas the control epidermis failed to exhibit any
suprabasal p63 staining (Fig. 2c
). Lines D1593 and D1595
also showed p63 staining in suprabasal nuclei (data not shown).
Effects of ML.Np63 Transgene Expression in the Epidermis.
The ML.Np63 transgenic epidermis appeared to develop normally,
with no macroscopically detectable difference between transgenic and
nontransgenic littermates (data not shown). Immunofluorescence analysis
of epidermal differentiation markers, keratin 1 and loricrin, revealed
no overt alteration in epidermal differentiation (data not shown). To
determine whether there was an alteration of the epidermal
proliferation rate, incorporation of BrdUrd was examined. There was no
statistically significant difference between the transgenic and
nontransgenic newborn epidermal proliferation rates (Fig. 3
).
|
Np63 Altered UV-B-induced Apoptosis.Np63 decreased in normal skin after UV-B
exposure, we examined the effect of forced expression of this isoform
on the UV-B response of the epidermis. After exposure to 100
mJ/cm2
UV-B, apoptotic keratinocytes were
detected using TUNEL analysis. We found that the ML.Np63
transgenic epidermis was less susceptible to UV-B-induced apoptosis
than the nontransgenic epidermis. As shown in Fig. 4a
, there are fewer TUNEL-positive nuclei in the transgenic
epidermis than in the nontransgenic littermate. All three ML.Np63
lines exhibited a statistically significant decrease in the number of
apoptotic keratinocytes after UV-B exposure (Fig. 4b
).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofREFERENCES |
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Np63, is dramatically
reduced at both the transcript and protein levels in response to UV-B.
This was intriguing because the p53 protein is stabilized
posttranscriptionally in response to UV-B. We generated a transgenic
mouse model to assess the effect of forced overexpression of Np63
in the epidermis, particularly in response to UV-B. Although the
ML.Np63 epidermis appeared to develop normally, the ability of
epidermal keratinocytes to undergo apoptosis in response to UV-B
exposure was compromised, highlighting the importance of
down-regulating this p63 isoform in response to UV-B.
The observation that the ML.Np63 transgenic epidermis appeared to
develop normally with an unaltered proliferation rate was not
completely surprising because it has been shown that overexpression of
Np63 alone in cultured cells that were not stressed by
DNA-damaging agents did not result in apoptosis (13)
. The
need to down-regulate Np63 after UV-B exposure raises questions
regarding p63 function in the normal versus stressed
epidermis. The Np63 isoform fails to transactivate a
p53-responsive reporter construct; however, other p63 isoforms that
contain the 5' region homologous to the p53 transactivation domain have
been shown to activate p53-responsive promoters such as the p21/WAF1,
BAX, and MDM2 promoters (13
, 21) . Additionally, at least
one p63 isoform, Np63, has been shown to interact directly with
p53-binding sites in an electrophoretic mobility shift assay
(13)
. Because this p63 isoform has the exact same DNA
binding domain as the Np63 isoform, it is not unreasonable to
think that the Np63 isoform may also interact directly with
p53-binding sites. Furthermore, the Np63 isoform has been shown
to suppress p53 transcriptional activity (13)
. Whether
this suppression occurs via direct protein-protein interaction or via
promoter binding competition remains unclear. However, it has been
shown that the various p63 isoforms can form oligomeric complexes among
themselves (13
, 22)
but fail to interact directly with p53
or a p53 mutant in in vitro assays. These results imply that
p63 is affecting the ability of p53 to transactivate target genes via
an indirect mechanism such as competition for DNA binding sites.
This report is the first to assess the functional consequences of deregulated p63 in the epidermis of transgenic mice. It was proposed that p63 plays an essential role in maintaining the proliferative capacity of the basal cells of stratified epithelia (13 , 23) , a scenario that the results presented here support nicely. If activation of p53 results in cell cycle arrest or cell suicide, then maintaining a situation in which p53 is not allowed to function, i.e., rapid p53 turnover or blocking target promoter binding sites, is important in the proliferative basal compartment. Perhaps in addition to altering the ability of p53 to transcribe its targets, p63 may control other genes that function in excluding p53 from the nucleus or contribute to the rapid turnover of p53 in the cytoplasm. When a cell incurs DNA damage, p63 is down-regulated, allowing p53 to step into action, protecting the cell from perpetuating damaged DNA. Deregulated p63 expression would interfere with this protective role of p53, possibly by competing with p53 for binding to DNA targets and/or exclusion from the nucleus and degradation via the actions of newly synthesized p63.
There are many questions that remain to be explored regarding the
function of p63 in both the unperturbed state as well as in response to
DNA-damaging events. Defining a role, if any, for p63 in epithelial
tumorigenesis awaits further analysis. Interestingly, Osada et
al. (10)
report mutations in the p63 DNA binding
domain in three epidermal carcinomas. Additionally, a syndrome known as
EEC (ectrodactyly, ectodermal dysplasia and facial clefts) has been
linked to germ-line mutations in p63, primarily within the DNA-binding
domain (24)
. Amplification of chromosome 3q, the
chomosomal location of the p63 gene, has been observed in
squamous cell carcinomas and advanced cervical carcinomas, supporting a
role for deregulated p63 expression in epithelial tumorigenesis
(25, 26, 27)
. This ML.Np63 transgenic model will provide
a useful in vivo system for further assessing the role of
p63 in the epidermis and in epidermal tumorigenesis, both UV-B-induced
and chemically induced tumorigenesis.
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Note Added in Proof |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofREFERENCES |
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Np63 isoforms in squamous cell carcinoma were
published (28
, 29)
.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Molecular and Cellular Biology, Baylor
College of Medicine, One Baylor Plaza, Houston, TX 77030. 
2 The abbreviations used are: K14, keratin 14;
RT-PCR, reverse transcription-PCR; RPA, RNase protection assay; ML,
mouse loricrin; BrdUrd, bromodeoxyuridine.
Received 4/18/00. Accepted 6/15/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofREFERENCES |
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M. S. Iordanov, R. J. Choi, O. P. Ryabinina, T.-H. Dinh, R. K. Bright, and B. E. Magun The UV (Ribotoxic) Stress Response of Human Keratinocytes Involves the Unexpected Uncoupling of the Ras-Extracellular Signal-Regulated Kinase Signaling Cascade from the Activated Epidermal Growth Factor Receptor Mol. Cell. Biol., August 1, 2002; 22(15): 5380 - 5394. [Abstract] [Full Text] [PDF]
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