From the
Human involucrin (hINV) is a cornified envelope precursor that
is specifically expressed in the suprabasal epidermal layers. We
previously demonstrated that 2500 base pairs of the hINV gene upstream
regulatory region confers differentiation appropriate regulation in
transgenic mice. An analysis of the hINV gene sequence upstream of the
transcription start site reveals five potential AP1 binding sites
(AP1-1 to 5). Using reporter gene constructs in human
keratinocytes, we show that the most distal (AP1-5) and most
proximal (AP1-1) AP1 sites are essential for high level
transcriptional activity. Simultaneous mutation of these sites reduces
transcription by 80%. Gel supershift experiments indicate the
interaction of these sites with Fra-1, junB, and
junD. Involucrin mRNA levels increase 10-fold and promoter
activity 5-11-fold when differentiation is induced by phorbol
ester. Functional studies implicate AP1-1 and AP1-5 in
mediating the phorbol ester-dependent increase in promoter activity. No
involucrin promoter activity or involucrin mRNA was detected in 3T3
fibroblasts. We conclude that (i) two AP1 sites in the hINV promoter
are important elements required for keratinocyte-specific expression,
(ii) these AP1-1 sites mediate the phorbol ester-dependent
increase in promoter activity, and (iii) Fra-1, junB,
and junD may be important regulators of hINV expression in
epidermis.
The human epidermis is a keratinizing squamous epithelium
consisting of several distinct layers (for review, see Ref. 1). The
basal layer is located adjacent to the dermis and consists of
relatively undifferentiated, proliferative cells. During the process of
terminal differentiation the keratinocytes, responding to unknown
stimuli, withdraw from the cell cycle, migrate from the basal to the
superficial layers of the epidermis, and simultaneously undergo
morphological and biochemical changes. The terminal cell consists of a
network of cytokeratin filaments surrounded by an insoluble envelope of
heavily cross-linked protein
(2, 3) . The envelope
components and transglutaminase, the enzyme responsible for assembly,
must be expressed at the proper time and level during differentiation
for the envelope to be formed
properly
(1, 4, 5, 6, 7, 8, 9) .
One of these components expressed specifically in the suprabasal layers
of the epidermis is involucrin. Involucrin is a highly reactive,
soluble, transglutaminase substrate, which functions as a glutamyl
donor during assembly of the cornified
envelope
(4, 6, 10, 11, 12, 13, 14, 15) .
Little is known about mechanisms responsible for tissue- and
differentiation-specific regulation in most systems. In skin, however,
evidence implicating the protein kinase C-AP1 pathways is rapidly
accumulating
(16, 17, 18) . AP1 was initially
described as a DNA binding activity in HeLa cell extracts that
recognized specific sites in the SV40 enhancer
(19) . Specific
DNA binding sites have since been identified in a wide variety of
genes
(20, 21) . The term AP1 describes a broad and
fairly ubiquitous class of transcription factors that consist of either
a homo- or heterodimer of two jun family proteins, or a
heterodimer of one jun and one fos family
protein
(22) . Three members of the jun family,
c-jun, junB
(23) , and
junD
(24, 25) have been identified, while the
fos family includes c-fos, fosB
(26) ,
fosB2, Fra-1
(27, 28) , and
Fra-2
(29, 30) . These proteins appear to be
expressed differentially in various cell types, can form heterodimers
with each other, and bind the same DNA consensus sequence. However,
they respond differently to activating stimuli (31-34), and they
appear to have differing effects on cell function (30, 35).
Several
findings have lead to speculation about a possible regulatory role for
AP1 proteins in the epidermal differentiation process. In most
quiescent adult human tissues, levels of the AP1 component
c-fos, for example, are extremely low, but are rapidly induced
by mitogenic stimuli
(36) . In contrast, fos is
constitutively expressed at high levels in the non-proliferative
differentiating suprabasal epidermal layers
(16, 18) . In
addition, AP1 has recently been implicated in the regulation of several
keratinocyte genes
(37, 38, 39) .
Transgenic
studies from our laboratory suggest that a 2.5-kilobase fragment of the
human involucrin gene (hINV)
To create the
plasmids containing mutated AP1-1 and/or AP1-5 sites (see
below, and ), convenient restriction sites flanking the AP1
sites were identified, and the fragments containing the wild type AP1
sites were released by restriction digestion. Oligonucleotides
identical to the wild type sequences between these restriction sites,
except for the mutated bases, were synthesized and ligated into the
parent constructs. Conservative thymidine to cytosine mutations at
position 5 of the consensus binding sequence were chosen
(43) .
It is important to note that the junction between the hINV gene
sequences and the luciferase reporter gene sequence are identical in
all constructs.
Fig. 4
shows a
gel mobility shift experiment in which keratinocyte nuclear extract was
incubated with
The AP1 Sites AP1-1 and AP1-5 Enhance hINV Gene
Expression-We have previously shown that 2.5 kilobase pairs of
hINV gene upstream regulatory region are sufficient to drive
tissue-specific expression in transgenic mice
(40) . In the
present study we have evaluated the ability of this region to drive
transcription in keratinocytes. Sequential truncation of the upstream
sequence results in a progressive loss of reporter gene activity. We
have identified five AP1 consensus binding sites in this region. Two
large drops in transcriptional activity are associated with the loss of
DNA segments containing the AP1-1 and AP1-5 sites
(Fig. 1). In contrast, the sites AP1-2, -3, and -4 make
less of a contribution to the overall promoter activity. However, each
deletion was quite large, potentially removing other unidentified
transcription factor binding sites (see Fig. 1). We therefore
confirmed by point mutation that AP1-1 and AP1-5 were
required for optimal activity. An 80% reduction in transcriptional
activity in keratinocytes was observed when AP1-1 and AP1-5
were simultaneously mutated, suggesting the these sites were necessary
for hINV expression in keratinocytes
(40) . Simultaneous mutation
of both sites does not, however, completely abolish promoter activity,
suggesting that other transcription factors mediate at least 20% of the
activity.
In summary, our results indicate that the
AP1-1 (distal promoter) and AP1-5 (proximal promoter)
sites, (i) function as general enhancer elements, and (ii) mediate the
differentiation-dependent increase in promoter activity in response to
phorbol ester.
AP1-1, -2, -3, -4, and -5 are the AP1 binding sites from the
hINV gene sequence with flanking sequence (see positions in Fig. 1).
AP1-1m, AP1-2m and AP1-5m are mutants of AP1-1,
-2, and -5 in which the T at position 5 of the consensus sequence is
changed to C (underlined in each oligonucleotide). AP1c, SP1c, and AP2c
contain the AP1, SP1, and AP2 consensus binding sites flanked by random
sequence (Promega). The length of each double stranded oligonucleotide
is shown at the right, the sequence of the putative AP1, SP1, or AP2
binding sites are indicated in bold.
(
)
upstream of the
transcription start site contains all of the information required for
tissue-specific and differentiation-appropriate targeting to the
epidermis
(40) . In the present study we (i) identify five AP1
sites within this upstream regulatory region and confirm that two of
these sites are necessary for expression of the gene in keratinocytes,
(ii) demonstrate that the promoter is inactive in fibroblasts, (iii)
show that the AP1 transcription factors Fra-1, junB,
and junD are major contributors to the protein complexes which
bind at these two sites, and (iv) show that these sites are important
mediators of the inductive response observed following treatment with
phorbol esters.
Chemicals and Reagents
Serum-free keratinocyte
media (KSFM), trypsin, Hanks' balanced salt solution, gentamicin,
and Lipofectin were obtained from Life Technologies, Inc. The plasmids
pGL2-Basic, pGL2-Control, and pSV--galactosidase, the GL-2
polymerase chain reaction primer, AP1 and SP1 consensus sequence
oligonucleotides, and the chemiluminescent luciferase assay systems
were obtained from Promega. The T/A cloning vector, pCRII, and
competent INV-
F` bacteria were purchased from Invitrogen. The
Galactolight
-galactosidase chemiluminescent assay system was
obtained from Tropix. Dispase was obtained from Boehringer Mannheim,
[
-
P]ATP from DuPont NEN. Phorbol ester
(12-O-tetradecanoylphorbol-13-acetate, TPA) and dimethyl
sulfoxide were from the Sigma. Chemiluminescence was measured using a
Berthold luminometer and oligonucleotides were synthesized on an
Applied Biosystems DNA synthesizer. Transcription factor binding sites
in the hINV promoter were identified using the Signal Scan DNA sequence
analysis program
(41) .
Antibodies
Antibodies against transcription
factors were purchased from Santa Cruz Biochemicals and were rabbit
polyclonal antibodies raised against synthetic peptides. The broad
spectrum anti-c-fos antibody (pan-fos) was raised
against the highly conserved c-fos amino acids 128-152,
and reacts with c-fos, fosB, Fra-1, and
Fra-2. The broad spectrum anti-c-jun antibody
(pan-jun) was raised against amino acids 247-263 of
mouse c-jun, and cross-reacts with c-jun,
junB, and junD. The remaining antibodies used in this
study (c-fos, fosB, Fra-1, Fra-2,
c-jun, junB, and junD) were raised against
sequences specific to each protein and do not cross-react.
Plasmid Construction
The plasmids used in this
study include pSP64I-3 H/Hc, which contains a
HindIII/HincII fragment of the hINV gene including
approximately 2.5-kilobase pairs of upstream regulatory sequence, the
TATA box, and the hINV intron
(12) , pUC1813
(42) , and
pGL2-Basic, a promoterless luciferase reporter vector. To construct
plasmids for the deletion series, a 2.4-kilobase pair
HindIII/CelII fragment of the hINV gene including
positions -7 to -2473 relative to the transcription start
site was released from pSP64
I-3 H/Hc. The ends of this fragment
were made blunt with Klenow polymerase prior to cloning into
SmaI-restricted pUC1813 to produce pUC1813
I-3
HindIII/CelII, in which the hINV segment is flanked
by BamHI sites. The BamHI fragment encoding the hINV
sequence was then subcloned into BglII-restricted pGL2-Basic.
The resulting clones were screened for the appropriate orientation of
the inserted DNA fragment by restriction mapping. The plasmid
containing the hINV insert in the appropriate orientation was named
pINV-2473 based on the nucleotide at the most 5` end of the hINV
segment, as determined by counting backwards from the start site of
transcription of the hINV gene
(12) . Maps of the hINV
promoter-luciferase fusion constructs are shown in Fig. 1. The
other plasmids in the series were produced by removing promoter
segments using restriction enzymes, or by reconstructing gene segments
using the polymerase chain reaction, and are also named according to
the 5` most nucleotide from the hINV sequence.
Figure 1:
Human involucrin promoter-luciferase
reporter gene fusion constructs. In each map, the narrow line represents the hINV sequence and the dark box symbolizes
the luciferase gene. The arrow indicates transcription of the
luciferase product. The plasmids are named (pINV-2473, pINV-2216, etc.)
based on the length of the segment, as measured backwards from the hINV
transcription start site. The five AP1 sites present within the
upstream sequence are numbered 1 to 5 and indicated by
circles. The scale at the top is in base
pairs.
pINV-793 was used as
a template to produce three constructs, pINV-679, pINV-489, and
pINV-298, by polymerase chain reaction. Involucrin sequence-specific
oligonucleotides served as upstream primers and a pGL2-Basic-specific
primer, GL-2 (Promega), was used as the downstream primer. The
polymerase chain reaction products were cloned into pCRII for further
amplification and finally subcloned into pGL2-Basic.
Tissue Culture
Primary human keratinocytes were
cultured from human foreskin samples. Foreskin specimens were stored
overnight at 4 °C in KSFM containing 5 µg/ml gentamicin. The
specimens were rinsed for several minutes in Hanks' balanced salt
solution containing 20 µg/ml gentamicin, cleaned of connective
tissue, and incubated for 18 h at 4 °C in Hanks' balanced
salt solution containing 10 mg/ml dispase to separate the epidermis
from the dermis
(44) . The sheet of epidermis was then
dissociated with 0.25% trypsin in Hanks' balanced salt solution
containing 1.0 mM EDTA for 5 min at 37 °C. The trypsin was
neutralized with serum and the cells were plated in KSFM at 1-3
10
cells per 10-cm
dish. The cells were
grown at 37 °C in a 5% CO
atmosphere, split at a 1:5
ratio when 70% confluent, and used for transfection at the third
passage. Mouse embryonic fibroblast 3T3 cells were grown as described
previously
(45) .
Transfection
Cells were transfected in 60-mm
diameter dishes when 60% confluent. Sixteen micrograms of Lipofectin
reagent, 3.5 µg of test plasmid, 0.5 µg of the
pSV--galactosidase (an SV40 promoter/enhancer
-galactosidase
reporter vector, as an internal standard) were mixed, added to cells in
3 ml of KSFM, and incubated for 5 h at 37 °C. A parallel set of
dishes was co-transfected with 3.5 µg of pGL2-Control (luciferase
driven by the SV40 promoter/enhancer, Promega) and 0.5 µg of
pSV-
-galactosidase. At 5 h, 3 ml of KSFM was added and the
incubation continued for an additional 19 h. The cells were then
allowed to recover for 24 h in fresh KSFM. At this point, the cultures
were washed once with KSFM and then treated for 24 h with KSFM
containing 50 ng of TPA/ml (from 5 mg/ml stocks in Me
SO) to
induce differentiation
(46) .
Luciferase Assay
The cells were washed twice with
phosphate-buffered saline, dissolved in 250 µl of cell culture
lysis reagent (Promega), and harvested by scraping. Luciferase assays
were performed immediately using a Berthold luminometer and the Promega
luciferase assay kit. Cell extract (20 µl) and 100 µl of
luciferin mixture were reacted for 5 s and light output was monitored
over the next 10 s. -Galactosidase assays were performed on
duplicate aliquots using the Galactolight reagent system. All assays
were performed in triplicate, and each experiment was repeated a
minimum of three times.
Normalization of Luciferase Activity
Within each
experiment, differences in transfection efficiency were normalized to
the -galactosidase internal standard by dividing the involucrin
luciferase signal by the pSV-
-galactosidase signal. However, use
of the
-galactosidase signal was not adequate to normalize between
experiments using different cell lines. For this reason, we divided the
normalized luciferase activity of each involucrin construct by the
normalized pGL2-Control luciferase activity and expressed the results
in dimensionless, arbitrary units.
Preparation of Nuclear Extracts
Passage 3
keratinocytes were grown in KSFM until 80% confluent and were then
treated with KSFM supplemented with 50 ng of TPA/ml for 24 h. Nuclear
extracts were prepared at approximately 0.5-2.0 mg of protein/ml
according to the method of Dignam et al.(47) or as
described by Schreiber et al.(48) .
Gel Mobility Shift Assay
Band shifts were
conducted by incubating a 20-µl reaction containing 15% glycerol,
75 mM KCl, 0.375 mM dithiothreitol, 0.375 mM
phenylmethylsulfonyl fluoride, 12.5 mM NaCl, 0.1 µg/µl
poly(dI-dC), 2.5 µg of nuclear extract, and 0.3 ng of radiolabeled
DNA for 5 min at room temperature. The samples were immediately
electrophoresed at 250 V for 1.5 h on 5% nondenaturing acrylamide gels
using a one-fourth TBE running buffer, dried, and
autoradiographed. For competition studies, radioinert DNA competitor
was added at a 20- or 200-fold molar excess.
Gel Mobility Supershift
The reaction mixtures were
assembled as outlined above without the P-labeled oligomer
and incubated with antibodies specific for jun or fos family members (1 µg of antibody per 2 µg of nuclear
extract) for 2 h at 4 °C. The
P-labeled DNA oligomer
was then added, incubated for 5 min at room temperature and then
immediately electrophoresed on a 5% nondenaturing acrylamide gel. In
control reactions, the antibody was replaced with bovine serum albumin
(1 µg of bovine serum albumin/2 µg of nuclear extract).
hINV Promoter Deletion Series
The constructs
shown in Fig. 1were transfected into cultured human epidermal
keratinocytes, and assayed for the ability to drive production of
luciferase activity (Fig. 2). pINV-2473 and pINV-2216 produce
high levels of luciferase activity. Elimination of 80 base pairs of
pINV-2136 reduces the activity by 45% and luciferase activity
fluctuates between 30 and 55% of maximum for pINV-1336, -1261, -1152,
-1091, -986, -793, -679, -489, -298, -241, and -159. However,
truncation to position -41, pINV-41, results in a drop in
activity to 2.5% of the level observed for plasmids pINV-2473 or
pINV-2216.
Figure 2:
Transcriptional activity of the intact
hINV promoter. Passage three keratinocytes were grown until 60%
confluent, then cotransfected with each hINV-luciferase test plasmid
and an internal control plasmid, pSV--galactosidase, as described
under ``Materials and Methods.'' Twenty-four hours after
transfection, the cells were treated for 24 h with fresh KSFM
containing 50 ng of TPA/ml. Extracts were prepared and assayed for
luciferase and
-galactosidase activity. hINV-luciferase activity
was normalized using
-galactosidase as an internal standard. The
normalized luciferase activity is expressed in arbitrary units as
defined under ``Materials and Methods.'' The x axis
measures the length of the hINV DNA segment in base pairs. The data
points from left to right correspond to the constructs shown in Fig.
1.
Location of AP1 Sites in hINV Promoter
The
upstream regulatory region of the hINV promoter contains four motifs
having homology to the AP1 binding site,
5`-TGANT(C/A)NN-3`, where N is any
nucleotide
(20) and one site similar to the putative AP1 motif
5`-GAGAGGAA-3`
(49) . Their positions in the hINV upstream
segment are shown in Fig. 1and their sequence in .
In the deletion series, the drop in activity observed between
-2216/-2136 and -159/-41 in
Fig. 2
(open circles) corresponds to the loss of
AP1-5 and AP1-1, respectively. Removal of AP1-2, -3,
or -4 does not appear to significantly influence the promoter activity.
hINV Promoter Point Mutations
To more specifically
analyze the contribution of AP1-1 and AP1-5 to the overall
activity of the hINV promoter, we generated hINV promoter constructs in
which the AP1-1 and/or AP1-5 site were inactivated
(i.e. rendered unable to bind AP1) by changing the T at
position 5 in the consensus sequence to C ()
(43) .
As shown in Fig. 3A, the mutation of AP1-1 reduces
the promoter activity (solid symbols) for each construct
tested.
Figure 3:
AP1-1, AP1-5, and hINV
promoter activity. In panel A, constructs pINV-2473, pINV-986,
pINV-793, pINV-298, pINV-241, and pINV-159 (data points left to right)
were modified to mutate the AP1-1 site by replacing sequence
AP1-1 with AP1-1 m (see Table I). The x axis
measures the length of the hINV DNA segment in base pairs and the y axis measures arbitrary units of luciferase activity. The open
square connected to each curve by a dotted line indicates
the pINV-41, the basal promoter, which lacks an AP1 site. It is
included to indicate the activity of the minimal hINV transcription
unit. In panel B, construct pINV-2473 was modified to
eliminate AP1-1, AP1-5, or AP1-1 and AP1-5 by
replacing these sites with AP1-1m, AP1-5m, or AP1-1m
and AP1-5m, respectively. Constructs were tested for activity by
transfection into normal human keratinocytes which were then treated
exactly as outlined as described in the legend to Fig.
2.
Similarly, to evaluate the role of AP1-5, cells were
transfected with construct pINV-2473 (see Fig. 1) containing an
intact or mutated AP1-5 site. Activity of this construct was
compared to the activity of a pINV-2473 AP1-1 site mutant and an
AP1-1/AP1-5 dual mutant (Fig. 3B). In the
experiment shown in Fig. 3B, elimination of AP1-1
or AP1-5 resulted in a 60 and 20% decrease in activity,
respectively. Elimination of AP1 consistently resulted in a greater
loss of promoter activity than elimination of AP1-5; however, the
actual percentages varied among keratinocyte strains. Simultaneous
mutation of AP1-1 and AP1-5 results in an 80% decline in
activity, suggesting that the contribution of each site to overall
activity is additive. Interaction of Nuclear Proteins with the AP1-1 and AP1-5
Sites-To characterize the interaction of nuclear proteins with
the AP1-like consensus sites in the hINV regulatory region, we
performed gel shift and oligonucleotide competition experiments. The
sequence of AP1-1 and -5 are shown in , along with
the sequence of other oligonucleotides used in the gel shift
experiments. AP1c is a commercially available oligonucleotide. The AP1c
consensus binding sequence is identical to the involucrin sequence, but
the flanking sequences are different. AP1-1m, AP1-2m, and
AP1-5m are mutants of the hINV AP1-1, AP1-2, and
AP1-5 sites, respectively, in which the T at position 5 has been
changed to C. The change of T to C at this position has been reported
to result in a loss of AP1 binding
(43) .
P-AP1-1 in the presence or absence of
excess radioinert AP1-1, AP1-1m, AP1c, SP1c, AP1-2,
or AP1-2m competitor. The
P-AP1-1
oligonucleotide shifts a major band (arrow) that is competed
by oligonucleotides AP1-1, AP1c, and AP1-2, but not by
AP1-1m, SP1c, or AP1-2m. Additional competition experiments
with
P-AP1-1 and increasing amounts of radioinert
AP1 indicate half-maximal competition at 20-50-fold molar excess
(not shown).
Figure 4:
Interaction of nuclear proteins with the
AP1-1 binding site. P-AP1-1 (0.3 ng) was
incubated with nuclear extract in the presence of 0, 20-, or 200-fold
molar excess of AP1-1, AP1-1m, AP1c, SP1c, AP1-2, or
AP1-2m. The incubations were then electrophoresed on
nondenaturing gels, dried, and autoradiographed with intensifying
screens at -70 °C. The arrow indicates the major
AP1-1 binding activity. N indicates a lane in which
nuclear extract was absent. The free (unbound) oligonucleotide is not
shown in this experiment.
A mobility shift assay in which
P-AP1-5 was incubated with radioinert AP1-5,
AP1-1, AP1-1m, AP1-2, AP1-2m, SP1c, and AP2c is
shown in Fig. 5. The results show specific binding of a major
band (arrow) that is competed by AP1 site-encoding
oligonucleotides (i.e. AP1-5, AP1-1, AP1-2,
and AP1c), but not by non-AP1-encoding sequences (i.e. SP1 and
AP2c). Some competition was observed when AP1-1m was present at a
200-fold molar excess, but a 200-fold molar excess of AP1-2m did
not compete. Identity of Nuclear Proteins Which Bind to AP1-1 and
AP1-5-To identify the factors that interact with the
AP1-1 site, nuclear extracts were incubated with
P-AP1-1 in the presence or absence of antibodies
specific for various jun and fos family members
(Fig. 6A). Lane N shows the migration of free
P-AP1-1 (arrowhead near gel front) in the
absence of extract and the minus lane indicates the shifted protein-DNA
complex observed in the absence of antibody (long arrow). The
broadly reactive jun and fos antibodies (pan), as
well as antibodies specific for fos family member
Fra-1 (F1) and jun family members
junB (B) and junD (D), produced
supershifted bands (short arrows). No shift was observed in
samples treated with antibodies specific for c-fos (c), fosB (B), Fra-2
(F2), or c-jun (c). An identical pattern of
supershifts is observed when AP1-5 is used as the radioactive
probe (Fig. 6B), suggesting that Fra-1,
junB, and junD are factors that bind to both
AP1-1 and AP1-5.
Figure 5:
Interaction of nuclear proteins with the
AP1-5 binding site. P-AP1-5 (0.3 ng) was
incubated with nuclear extract in the presence of a 0, 20-, or 200-fold
molar excess of AP1-5, AP1-1, AP1-1m, AP1-2,
AP1-2m, AP1c, SP1c, or AP2c. The incubations were electrophoresed
on nondenaturing gels, dried, and autoradiographed. The arrow indicates the major AP1-5 binding activity, the
arrowhead indicates free
P-AP1-5 and N indicates a lane in which the nuclear extract was
omitted.
Figure 6:
Identity of nuclear factors that interact
with the hINV promoter AP1-1 and AP1-5 sites. Nuclear
extracts were incubated with 0.3 ng of P-AP1-1
(A) or 0.3 ng of
P-AP1-5 (B) in
the absence (-) or presence of antibodies against pan-fos (pan), c-fos (c), fosB
(B), Fra-1 (F1), Fra-2
(F2), pan-jun (pan), c-jun (c), junB (B), or
junD (D). The complexes were then electrophoresed on
a 5% nondenaturing acrylamide gel. The long arrows to the
left in each panel indicate the complex formed in the absence
of antibody. The short arrows to the right indicate
the supershifted complexes. The arrowhead near the gel front
indicates free
P-AP1 oligomer. Nuclear extract was omitted
in the lane marked N.
Phorbol Ester-dependent Induction of hINV Gene
Expression
As shown above, the AP1-1 and AP1-5 sites
are functionally important in maintaining optimal transcription in
cultures grown in the presence of the differentiating agent, TPA.
Fig. 7
shows that TPA treatment (+) increases the level of
AP1 activity which binds to the hINV AP1-5 site (and AP1-1, not
shown) compared to nuclear extracts prepared from untreated(-)
cultures. The level of AP1 binding activity increased between 25 and
200-fold depending upon the experiment. As AP1 has been implicated in
mediating the effects of phorbol esters in a variety of
systems
(20) , this suggested that these sites may mediate the
TPA-dependent induction of hINV mRNA expression that has been
previously reported in keratinocytes
(50) . We therefore further
investigated the role of AP1-1 and AP1-5 by testing the
activity of full-length (pINV-2473) constructs in which one or both
sites are inactivated by point mutation. As summarized in Fig. 8,
mutation of the AP1-1, AP1-5, or both sites simultaneously
reduces both basal and TPA-stimulated transcriptional activity. In the
case of mutation of AP1-5, the overall activity is decreased, but
the fold induction by TPA remains stable. Inactivation of AP1-1,
in contrast, results in a decrease in the fold induction following TPA
treatment. Simultaneous inactivation of AP1-1 and AP1-5
resulted in a >50% reduction in the TPA response compared to the
intact construct.
Figure 7:
Phorbol ester increases AP1 binding to AP1
DNA response elements. Nuclear extracts were prepared from cells grown
for 24 h in the presence (+) or absence (-) of 50 ng/ml TPA.
P-AP1-5 (0.3 ng) was incubated with each extract in
the presence or absence of 0, 20-, and 200-fold molar excess of
AP1-5 or AP1c. The extracts were then electrophoresed on
nondenaturing gels, dried, and autoradiographed with intensifying
screens at -70 °C. The bracket indicates migration
of the major AP1 binding activity. Nuclear extract was omitted in the
lanes marked N. The arrowhead indicates migration of
the free, unbound oligonucleotide.
Figure 8:
Mutation of AP1-1 sites reduces hINV
promoter response to TPA. Normal human keratinocytes were transfected
with pINV-2473 (the full-length hINV promoter, Intact), or with
variants in which AP1-5 (AP1-5m), AP1-1
(AP1-1m), or AP1-1 and AP1-5 (AP1-1m/5m) are
inactivated by point mutation. The cells were then treated in the
presence (+TPA) or absence (-TPA) of 50
ng/ml TPA for 24 h, harvested, and assayed for luciferase activity. All
activities are normalized by setting the activity of the intact
promoter in the absence of TPA to 100. The fold increase, X,
indicates the ratio of activity in the presence of TPA/activity in the
absence of TPA for each construct. The numbers at the top of the figure indicate the distance in base pairs along the hINV
promoter and the black box indicates the luciferase reporter
gene. The positions of AP1-1, -2, -3, -4, and -5 are indicated by
the five open circles.
Tissue Specificity of hINV Promoter Activity and Phorbol
Ester Response
As shown in Fig. 9, TPA significantly
increases the level of human involucrin mRNA present in keratinocytes.
In contrast, no basal expression and no TPA responsive induction of
involucrin mRNA are observed in NIH 3T3 cells. Promoter transfection
studies in keratinocytes indicate a 7.5-11-fold increase in
promoter activity following TPA treatment for the pINV-2473 and
pINV-241 constructs. In parallel experiments, no promoter activity and
no TPA response were observed in 3T3 cells (Fig. 9). These
results suggest that the promoter activity is restricted to
keratinocytes and requires the AP1-1 and AP1-5 sites.
Figure 9:
Phorbol ester regulation of involucrin
promoter activity and mRNA level. Normal human keratinocyte
(NHK) and 3T3 mouse fibroblast (3T3) cultures were treated in
the presence (+) or absence (-) of 50 ng/ml TPA for 24 h.
Poly(A) RNA was prepared, electrophoresed, and
transferred to Biodyne A membrane as described previously (62). The
membranes were hybridized with a
P-labeled cDNA probes
encoding human (NHK cells) or mouse (3T3 cells) involucrin
(INV). The loading was normalized by hybridizing with an actin
cDNA probe (A). Transcriptional activity of the human
involucrin promoter (constructs pINV-2473 and pINV-241) was monitored
as described under ``Materials and Methods'' and in the
legend to Fig. 2. The activity bars shown in the 3T3 assay
indicate background activity (i.e. the promoter was completely
inactive in this cell type). The SV40 promoter, transfected in parallel
as a positive transfection control, was active in both cell types. The
mouse involucrin cDNA was kindly provided by Drs. P. Djian and H. Green
(63).
hINV Promoter Is Silent in Fibroblasts
In contrast, the
hINV promoter was found to be completely inactive in fibroblasts. This
is consistent with the observation that no involucrin mRNA is detected
in fibroblasts
(12, 15, 40) (Fig. 9).
Thus, although AP1-1 and AP1-5 are necessary for activity
in keratinocytes, these sites are not sufficient to mediate
transcriptional activation in fibroblasts. Possible explanations for
the lack of activity of the hINV promoter in 3T3 cells is that 3T3
cells lack the necessary AP1 transcription factors and/or these factors
are not appropriately phosphorylated. However, AP1-binding proteins
have been described in fibroblasts
(51) . Alternatively, some
other factor may suppress hINV promoter transcription in 3T3 cells.
Moreover, AP1 itself has been reported to suppress transcription in
some systems
(52) . Finally, a cofactor may be required for
activation of hINV gene expression, expression of such a factor could
be restricted to keratinocytes.
Activator Protein-1 Is Necessary for
Differentiation-dependent Induction of hINV Expression
TPA has
been shown to increase keratinocyte differentiation and to modulate
gene expression in keratinocytes
(50) . AP1 has been implicated
in mediating the effects of phorbol esters in a variety of
systems
(20) . Our results show that TPA produces a
10-100-fold increase in AP1 binding to the hINV promoter
AP1-1 and AP1-5 sites. This is consistent with a role for
AP1 factors in mediating the differentiation-dependent increase in hINV
gene expression. It was therefore important to determine whether the
effects of TPA on hINV promoter activity were mediated by AP1. The
point mutation experiment, shown in Fig. 8, suggests that the TPA
response is partially mediated via the AP1-1 and AP1-5
binding sites. The fold induction is slightly reduced by mutation of
AP1-1 or AP1-5 and further reduced by simultaneous
inactivation of AP1-1 and AP1-5. These results suggest that
the AP1-1 and AP1-5 sites cooperate to mediate one-half of
the phorbol ester induction. This also implies that other transcription
factors must participate in the induction events. The possibility that
binding sites for these factors are localized in the hINV distal
promoter region is currently being investigated. Identity of fos and jun Family Members Binding to AP1-1 and
AP1-5-Gel mobility shift experiments demonstrate the
specific binding of nuclear proteins to oligonucleotides containing the
AP1-1 and AP1-5 sites. Competition for binding to
radiolabeled AP1-1 or AP1-5 oligonucleotides was observed
with oligonucleotides containing intact AP1-1 sites, but not with
oligonucleotides containing mutant AP1 sites. In addition,
oligonucleotides containing sites for other transcription factors
(i.e. SP1, AP2, etc.) did not compete. Antibody supershift
experiments using broad spectrum anti-fos and anti-jun antibodies (i.e. antibodies that react with all known
family members) strongly suggest the participation of both fos and jun family proteins in the formation of the complexes
which bind at the involucrin AP1-5 and AP1-1 sites. A more
detailed supershift analysis using antibodies specific for each family
member suggests that Fra-1 is the fos family
contributor and junB and junD the jun family
contributors. Fra-1 has been identified in heart and skeletal
muscle
(53, 54) , brain
(55) ,
leukocytes
(32) , fibroblasts (51), adipocytes
(56) , the
vascular system
(57) , and in skeletal tissues
(58) . This,
however, is the first study to support a regulatory function for
Fra-1 in keratinocytes. The 80% reduction in promoter activity
in the absence of AP1-1 and AP1-5 suggests that
Fra-1 may play a major role in the activation of hINV
expression in the suprabasal epidermal layers. Immunohistological
studies(
)
indicate that Fra-1 is
localized in the epidermal suprabasal layers. junB and
junD have been implicated in the keratinocyte-specific
expression of the human papillomavirus type 18 promoter
(59) .
Our results showing junB and junD binding to the hINV
promoter suggest that these factors are important for hINV expression
in keratinocytes.
Implications for Keratinocyte Differentiation
Studies
In the present study we show that the AP1 transcription
factor is necessary for expression of the hINV gene in epidermal
keratinocytes. AP1 is particularly interesting in the broader context
of a terminal differentiation program in which some genes are turned on
while others are concurrently turned off, since it can act as both an
activator and a repressor of transcription
(52) . Extracellular
calcium concentrations are higher in the upper than in the lower layers
of the epidermis in vivo. In vitro, similar elevations of
extracellular calcium cause keratinocytes to terminally differentiate
and form envelopes
(60, 61) . In keratinocytes, one
intracellular response to increased extracellular calcium is the
elevation of diacylglycerol levels and the activation of the protein
kinase C pathway
(61) and induction of terminal differentiation
in tissue culture systems by the diacylglycerol analogue TPA most
likely occurs by activation of protein kinase C. Protein kinase C is
known to influence gene expression through AP1. In most systems, the
activation of AP1 is a transient occurrence associated with mitogen
stimulation
(36) . In skin, however, the AP1 factor,
c-fos, is constitutively expressed in the suprabasal
layers
(16) , suggesting it may have a role in regulating
differentiation-specific gene expression. Our study suggests that hINV
expression in keratinocytes is enhanced by the combined activity of
several AP1 transcription factors, including Fra-1,
junB, and junD and implies that these factors may be
important for the differential regulation of expression of other
keratinocyte genes.
Table:
Sequence of hINV AP1 sites and oligonucleotides
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.