(Received for publication, June 6, 1994)
From the
Interferon- (IFN-
) has been shown to regulate
epidermal keratinocyte growth and differentiation. In this study, we
examined the effects of recombinant human IFN-
on the expression
of the gene encoding the 230-kDa bullous pemphigoid antigen (BPAG1), a
marker of the mitotic basal cell phenotype in the epidermis. Northern
analysis revealed a dose- and time-dependent suppression of BPAG1
expression by IFN-
in cultured human keratinocytes from several
different donors, and incubation of the cells with IFN-
in the
presence of cycloheximide demonstrated that this effect required
ongoing protein synthesis. The inhibition of BPAG1 gene expression was
also demonstrated at the protein level by indirect immunofluorescence
using a monoclonal antibody recognizing the human 230-kDa bullous
pemphigoid antigen. Transient transfections of cultured keratinocytes
with BPAG1 promoter-chloramphenicol acetyltransferase reporter gene
plasmids indicated marked suppression of the promoter activity by
IFN-
, and deletion constructs were able to identify a defined
region containing the responsive element (IFN-
inhibitory
element). Reduced transcription of the BPAG1 gene by IFN-
was also
demonstrated by in vitro nuclear run-on assays. These data,
which indicate inactivation of transcription of a basal
keratinocyte-specific gene (BPAG1) by IFN-
, provide novel insight
into the mechanisms of IFN-
-mediated keratinocyte gene regulation
and epidermal differentiation in inflammatory diseases.
Terminal differentiation of epidermal keratinocytes from the
proliferative basal phenotype to formation of the cornified envelope
involves activation and inactivation of a variety of genes at different
levels of the epidermis. One of the markers of the mitotic basal cell
phenotype is the expression of bullous pemphigoid antigens (BPA), ()two hemidesmosomal proteins that were initially recognized
as autoantigens in a blistering skin disease, bullous
pemphigoid(1, 2, 3) . It is now known that
there are at least two bullous pemphigoid antigens, the 230- and
180-kDa BPA; the corresponding genes are known as BPAG1 and BPAG2,
respectively(4, 5) . Recent cloning of BPAG1 and BPAG2
complementary and genomic DNA sequences has shown that these two
proteins are clearly distinct gene
products(6, 7, 8, 9, 10, 11, 12, 13) .
Recent studies have demonstrated that interferon- (IFN-
)
regulates keratinocyte growth and differentiation(14) . This
cytokine, a product of activated T cells and natural killer
cells(15) , has been shown to affect the expression of a
variety of genes(16) . In general, the IFN-
effects are
up-regulatory, and the corresponding transcriptional mechanisms have
been elucidated in the case of several
genes(16, 17, 18) . In selected cases, as for
example in the case of type I collagen, IFN-
down-regulates the
expression of the corresponding genes, COL1A1 and COL1A2, but the
mechanism is clearly post-transcriptional(17) .
IFN-
has two well documented effects on epidermal cell biology. First, this
cytokine induces the expression of intercellular adhesion molecule-1,
which serves as the binding molecule for lymphocyte function-associated
antigen-1, mediating lymphocyte-keratinocyte interactions(19) .
Furthermore, IFN-
induces the expression of HLA-DR on
keratinocytes(20, 21) . Thus, the effects of IFN-
play a key role in epidermal immune responses. Second, IFN-
has
direct inhibitory effects on keratinocyte growth and induces terminal
differentiation(14, 22) . Specifically, IFN-
up-regulates the expression of differentiation-specific genes,
including transglutaminase type I and a variety of proteins involved in
the formation of the cornified envelope(14) .
Since
down-regulation of BPA gene expression is one of the earliest events
during epidermal differentiation, in this study, we examined the
effects of IFN- on BPAG1 and BPAG2 gene expression.
The intensity of the autoradiographic bands was quantitated by scanning densitometry of the x-ray films at 633 nm, and the BPAG1 and BPAG2 mRNA steady-state levels were corrected by the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA transcripts in the same RNA preparations.
Figure 1:
Effects of IFN- on BPAG1 and BPAG2
gene expression in cultured human keratinocytes. The cells were
incubated with recombinant human IFN-
at concentrations varying
from 0 to 1000 units/ml for 24 h. Total RNA was then isolated, and
Northern analyses with BPAG1-, BPAG2-, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)-specific cDNAs were performed as
described under ``Materials and Methods.'' A,
autoradiograms of the Northern filters; B, quantitation of
relative mRNA levels by scanning densitometry. The values are expressed
as a percent of control cultures incubated in parallel without
IFN-
(100%).
Figure 2:
Time-dependent inhibition of BPAG1 gene
expression by IFN-. Cultured keratinocytes were incubated in the
presence of 100 units/ml IFN-
for the time periods indicated, and
BPAG1 and BPAG2 mRNA steady-state levels were determined by Northern
hybridizations as described in the legend to Fig. 1. A,
autoradiograms of the Northern filters; B, quantitation of the
mRNA levels by scanning densitometry. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
To examine whether the IFN--elicited
down-regulation of the BPAG1 gene was dependent of ongoing protein
synthesis, similar inhibition studies were performed in the presence
and absence of cycloheximide (10 µg/ml). As indicated above,
incubation of cultured keratinocytes with IFN-
(1000 units/ml) for
24 h in the absence of cycloheximide resulted in essentially complete
reduction of BPAG1 mRNA steady-state levels (Fig. 3). Incubation
of parallel cultures with cycloheximide alone resulted in
50%
inhibition of the BPAG1 mRNA steady-state level after correction for
the glyceraldehyde-3-phosphate dehydrogenase mRNA levels, which were
slightly increased in the presence of cycloheximide. However, the
addition of IFN-
failed to down-regulate the BPAG1 mRNA levels in
the presence of cycloheximide (Fig. 3). These observations
suggest that the effect of IFN-
on BPAG1 gene expression requires
ongoing protein synthesis, possibly induction of trans-acting
regulatory proteins.
Figure 3:
Demonstration that IFN--elicited
down-regulation of BPAG1 gene expression at the mRNA level is dependent
on ongoing protein synthesis. Keratinocyte cultures were incubated in
the presence (+) or absence(-) of IFN-
(1000 units/ml)
and/or cycloheximide (CHX; 10 µg/ml) for 24 h. Northern
hybridizations with BPAG1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were performed as described in the legend to Fig. 1.
Figure 4:
Selective inhibition of BPAG1 promoter
activity in transient transfections of cultured keratinocytes. Human
epidermal keratinocytes were transfected with the pBP2.6CAT construct,
which consists of human BPAG1 gene 5`-flanking DNA sequences extending
from positions -2536 to -1 linked to the CAT reporter gene.
The keratinocytes were cotransfected with Rous sarcoma
virus--galactosidase, which was used as an internal control of
transfection efficiency. The transfected cells were incubated with
IFN-
at concentrations varying from 0 to 1000 units/ml (lanes
1-4 in A). Parallel transfections were performed
with a thymidine kinase promoter-CAT construct in the absence or
presence of 1000 units/ml IFN-
(lanes1 and 2 in B). After 48 h of incubation, the CAT activity
was determined as described under ``Materials and Methods.''
The relative CAT activity was determined by counting the radioactivity
in the acetylated forms of radioactive chloramphenicol (AC) as
a percent of the total radioactivity in the acetylated and
non-acetylated (C) forms of
[
C]chloramphenicol used as
substrate.
To identify the region within the 2.6-kb BPAG1 promoter
that is responsive to IFN-, eight additional 5`-deletion
constructs were similarly tested in transient transfections. Each
construct was transfected in parallel to duplicate cultures, with or
without concomitant treatment with IFN-
(100 units/ml) (Fig. 5). The results indicated that seven of the constructs,
with their 5`-ends at positions -1806 to -216, which
demonstrated significant CAT activity, were responsive to IFN-
.
The construct extending from positions -176 to -1 had too
low level of expression to evaluate IFN-
response. Nevertheless,
the putative IFN-
inhibitory element resides within
0.2 kb of
the BPAG1 promoter region.
Figure 5:
Transient transfections with BPAG1
promoter 5`-deletion CAT constructs. Cultured keratinocytes were
transfected with constructs containing the BPAG1 promoter region with
their 5`-ends at the positions indicated. The cultures were then
incubated without(-) or with (+) IFN- (100 units/ml),
and the CAT activity was determined as described in the legend to Fig. 4.
To further examine the modulation of
BPAG1 gene expression by IFN- at the transcriptional level, in
vitro nuclear run-on transcription assays were performed.
Quantitation of the BPAG1 RNA transcripts in nuclei isolated from
keratinocytes, incubated with or without IFN-
(1000 units/ml),
indicated a 68% reduction by IFN-
after correction for ribosomal 7
S RNA transcripts in the same incubation (Fig. 6). No
hybridization was noted with the pBluescript vector as a negative
control (Fig. 6).
Figure 6:
In vitro nuclear run-on
transcription analysis. Isolated human epidermal keratinocyte nuclei
were incubated with [P]UTP, and radiolabeled RNA
transcripts were isolated as described under ``Materials and
Methods'' and elsewhere(31) . Human BPAG1 and ribosomal 7
S unlabeled cDNAs, as well as the pBluescript vector (BS), 5
µg each, were immobilized on nitrocellulose filters. The filters
were prehybridized and then hybridized with radiolabeled RNAs. Left, autoradiogram from hybridization with RNA from control
cell nuclei; right, nuclei from cells incubated with IFN-
(1000 units/ml) for 24 h.
Figure 7:
Demonstration of reduced 230-kDa BPA
protein levels in human keratinocytes incubated with IFN-.
Confluent cultures of epidermal keratinocytes were incubated for 0 or
24 h with 1000 units/ml IFN-
. The presence of the 230-kDa BPA
protein was determined by indirect immunofluorescence with a monoclonal
antibody.
In this study, we have demonstrated that IFN-
effectively reduces the expression of the 230-kDa BPA in cultured
keratinocytes as detected both at the mRNA and protein levels. This
novel finding contrasts with the induction of a variety of keratinocyte
gene products by IFN-
, including intercellular adhesion molecule-1
and HLA-DR(19, 20) . Furthermore, IFN-
is an
up-regulatory cytokine for the expression of a variety of genes at the
transcriptional level, and specific IFN-
-responsive elements, such
as the IFN-stimulated response element and
-activated site (see
below), have been identified in the promoter regions of these
genes(16, 18) . Mechanistically, the induction of
transcription by IFN-
involves activation of a latent cytoplasmic
factor,
-activated factor (GAF), by tyrosine
phosphorylation(34, 35) . Activated GAF is then
translocated into the nucleus and binds to a cis-element, the
-activated site, first recognized in the gene encoding a
guanylate-binding protein(34, 35, 36) . The
GAF protein was subsequently shown to be identical to a 91-kDa
DNA-binding protein, one of the four components of
interferon-stimulated gene factor-3, a transcription factor previously
shown to be activated by IFN-
(35) . The
interferon-stimulated gene factor-3 complex then recognizes the
interferon-stimulated response element present in a variety of
IFN-
-inducible
promoters(37, 38, 39, 40) . The
phosphorylation of GAF in response to IFN-
has been shown to be
mediated by kinases known as the Jak family of protein-tyrosine
kinases(41, 42) . In particular, Jak1 is activated in
response to both IFN-
and IFN-
, while Jak2 responds
exclusively to IFN-
(43) . Thus, these kinases are required
for nuclear signaling induced by IFN-
.
The reduced BPAG1 mRNA
steady-state levels, as demonstrated in this study, result from reduced
transcriptional activity of the gene, as shown by transient
transfection experiments and nuclear transcription assays. The
mechanisms of this down-regulation of gene expression are not clear,
but they are unlikely to involve direct binding of GAF or
interferon-stimulated gene factor-3 to DNA since ongoing protein
synthesis was required for the IFN- effect, as demonstrated by
cycloheximide experiments. Previously, IFN-
has been shown to
abrogate lipopolysaccharide-mediated enhancement of tumor necrosis
factor receptor expression in murine peritoneal
macrophages(44) . This effect also took place at the
transcriptional level, but the mechanism appears to be different from
our observations since the IFN-
effect was independent of ongoing
protein synthesis(44) .
The use of 5`-deletion constructs in
transient transfections indicated that the putative IFN-
inhibitory element resides within 0.2 kb of the upstream promoter
region of the BPAG1 gene. Examination of this sequence identified
keratinocyte-responsive element-3, which has been recently shown to
confer keratinocyte-specific expression to the BPAG1 gene(45) .
However, as indicated above, the keratinocyte-responsive element-3
sequence, 5`-CAAATATTTG-3` at positions -213 to -204, has
no similarity to previously published IFN-
-responsive elements.
Specifically, careful examination of the BPAG1 promoter region between
positions -216 and -1, which was shown to contain the
putative IFN-
inhibitory element, did not reveal a nucleotide
sequence reminiscent of the
-activated site consensus sequence,
TTNCNNNAAA (36) , or of the IFN-stimulated response element,
(G/A)GGAAAN(N)GAAACT (18) .
The observations presented in
this study provide novel insight into the mechanisms of keratinocyte
gene expression, indicating that IFN- is a potential inducer of
epidermal differentiation as reflected by down-regulation of a basal
cell-specific gene, BPAG1. The transcriptional down-regulation of the
BPAG1 gene was selective in that the expression of another
hemidesmosomal gene, BPAG2, was not affected by IFN-
as determined
at the mRNA steady-state level. Since BPAG2 is also expressed
exclusively in the basal keratinocytes within the epidermis,
differential mechanisms are required for down-regulation of BPAG1 and
BPAG2 genes at the suprabasilar level in vivo. These
observations attest to the complexity of epidermal differentiation
under physiological conditions.