(Received for publication, August 17, 1995; and in revised form, September 27, 1995)
From the
-Antitrypsin (
-AT) is one of
the major proteinase inhibitors in serum. Its primary physiological
function is to inhibit neutrophil elastase activity in lung, but it
also inhibits other serine proteases including trypsin, chymotrypsin,
thrombin, and cathepsin. We have previously reported a novel
-AT, S-2 isoform, from rabbit that is induced up to
100-fold in the liver during acute inflammatory condition (Ray, B. K.,
Gao, X., and Ray, A.(1994) J. Biol. Chem. 269,
22080-22086). Here, we present evidence that the expression of
this
1-AT S-2 gene is also induced in lipopolysaccharide
(LPS)-treated peripheral blood monocytes. From the cloned genomic DNA,
we have identified a distal LPS-responsive enhancer located between
-2438 and -1990 base pairs upstream of the transcription
start site. In vitro DNA-binding studies demonstrated an
interaction of an LPS-inducible NF-
B-like nuclear factor with a
B-element present in this enhancer region. Antibodies against p65
and p50 subunits of NF-
B supershifted the DNA-protein complex. A
mutation of the NF-
B-binding element virtually abolished the
LPS-responsive induction of the chimeric promoter in monocytic cells.
Furthermore, overexpression of NF-
B induced the wild-type promoter
activity. Taken together, these results demonstrated that during
LPS-mediated inflammation, NF-
B/Rel family of transcription
factors play a crucial role in the transcriptional induction of the
inflammation responsive
-AT gene.
-Antitrypsin (
-AT), (
)also known as
proteinase inhibitor, is
one of the major protease inhibitors in plasma.
-AT
has a broad range of activities (Laurell and Jeppsson, 1975) but mainly
protects the elastic fibers in lung alveoli from excessive digestion by
neutrophil elastase (Olsen et al., 1975; Gadek et
al., 1980). The importance of this particular function was first
proposed by Laurell and Eriksson(1963), after observing that
genetically
-AT-deficient individuals often develop
either a degenerative lung disease early in life (Eriksson, 1964) or a
liver disease (Sharp et al., 1969). Genetic
-AT deficiency seems to affect 1 in 2000 Europeans and
is manifested by emphysema in adults and liver disease in children. The
genetic variants associated with the deficiency of
-AT
are termed as P
, P
and
P
. Individuals homozygous for the
P
allele exhibit a deficiency level of 60%;
those homozygous for the P
allele exhibit
10-15% deficiency; and P
individuals
show almost no detectable amount of
-AT in their serum
(Gitlin and Gitlin, 1975; Allen et al., 1974; Muensch et
al., 1986). Increased expression of
-AT also have
clinical importance in the case of individuals carrying
P
-AT mutant allele. A
glutamate to lysine substitution at position 342 (Nukiwa et
al., 1987; Sifers et al., 1988) results in aggregation of
the mutant
-AT protein within the rough endoplasmic
reticulum (Lomas et al., 1992) of hepatocytes and forms
insoluble intracellular inclusions which lead to the hepatocellular
damage in these patients. There is now strong evidence that the liver
disease of the P
homozygotes is a direct
consequence of
-AT accumulation and degradation in the
hepatocyte (Carlson et al., 1988). Since inflammatory
condition increases the synthesis of
-AT protein
severalfold, prevention of inflammation and pyrexia could considerably
lower the accumulation of the defective protein in the liver of a
homozygous P
patient.
-AT
is primarily synthesized in the liver (Laurell and Jeppsson, 1975), to
a lesser extent in bronchoalveolar and breast milk macrophages, and in
blood monocytes (Perlmutter et al., 1985). It is also
reportedly expressed at lower levels in submandibular glands (Chao et al., 1990), renal tubular epithelial cells, intestinal
epithelial cells, nonparietal cells of gastric mucosa, and pancreatic
islet cells (Carlson et al., 1988; Kelsey et al.,
1987; Koopman et al., 1989). The plasma level of
-AT in a variety of species is normally 2-4
mg/ml, but levels increase 3-4-fold under inflammatory
conditions, pregnancy, and after administration of synthetic androgen
danazol (Laurell and Rannevik, 1979).
Due to the clinical
importance, molecular basis of the regulation of -AT
gene expression has been intensively studied. The minimal promoter
element required for liver specific basal expression of human
-AT gene was reported to be confined within 261
nucleotides from the transcription start site (Cilliberto et
al., 1985; DeSimone et al., 1987). But the mouse
-AT gene which is structurally similar to the human
gene requires 500 bases upstream of the transcription start site for
its maximal basal expression (Grayson et al., 1988). The
difference between the enhancers of these two genes is very surprising.
Also, the presence of negative elements that are active in liver cells
is reported for both human (DeSimone and Cortese, 1989) and mouse
-AT genes (Montgomery et al., 1990). Despite
numerous reports on
-AT promoter and its
liver-specific expression, very little is known regarding the
mechanisms or regulatory elements that control inducible expression of
-AT during inflammatory condition. It is only known
that, in liver,
-AT is induced by IL-6, whereas in
monocytes and macrophages it is induced by both IL-6 and bacterial
lipopolysaccharide (Perlmutter et al., 1989; Barbey-Morrel et al., 1987). During IL-6-induced expression, human
-AT promoter utilizes a distal transcriptional
initiation site located about 2 kb upstream of the hepatocyte-specific
transcription start site (Hafeez et al., 1992). A lack of
reports on the mechanisms of the inducible expression of
-AT is possibly due to its moderate level of induction
which makes it less conducive for such regulation studies. Our recent
studies have identified a highly inducible isoform of
-AT in rabbit whose expression is increased about
100-fold in response to inflammatory signals (Ray et al.,
1994). Rabbit genome contains multiple
-AT genes that
are significantly different in terms of their expression under
inflammatory condition. In response to inflammation, the mRNA level of
the two members of this family, F and S-1, increases nominally
(1.5-fold), whereas expression of the S-2 isoform increases about
100-fold. Thus S-2 isoform becomes a major component of
-AT in the rabbit under inflammatory conditions (Ray et al., 1994). We have therefore initiated a study to
understand the regulation of the inducible expression of the rabbit
gene as a prelude to the establishment of a rabbit model system to
study the pathology associated with the overexpression and deficiency
of
-AT.
To identify the elements required for
transcriptional induction of the rabbit -AT S-2 gene,
we performed a detailed analysis of the S-2 promoter region. In this
report, we show that a distal region located about 2.5 kb upstream of
the transcription start site confers the LPS inducibility to the S-2
promoter. We have identified one NF-
B-like-binding element (Sen
and Baltimore, 1986) at this region. Mutation of the NF-
B site
virtually eliminates LPS responsiveness in a reporter construct
containing the rabbit S-2 upstream promoter. Further characterization
showed that several members of the NF-
B family can bind to this
NF-
B enhancer motif. Also, cotransfection of NF-
B induces
expression of the reporter CAT construct. These results provide strong
evidence that NF-
B might be involved as a major regulator in the
induction of
-AT gene brought about by LPS-mediated
inflammation.
A palindromic
NF-B oligonucleotide sequence used as a competitor in EMSA is
5`-GATCCATGGGGAATTCCCCATG-3`. For self-annealing, the oligonucleotide
was heated at 95 °C for 2 min in 50 mM Tris, pH 7.4, 60
mM NaCl, 1 mM EDTA and allowed to cool slowly to room
temperature for 2-3 h. A mutant NF-
B oligonucleotide was
also used as a competitor in EMSA, and its sequence is
5`-GATCCATCTCGAATTCGAGATG-3`. Underlined bases represent mutated
sequences.
The BNL liver cell (BNL CL.2)
used in the transfection assays (ATCC TIB73) is a normal embryonic
liver cell line. These cells were cultured in Dulbecco's modified
Eagle medium containing a high level of glucose (4.5 g/liter) and
supplemented with 10% fetal bovine serum. Ten µg of reporter
plasmid were used in each transfection assay, with 2 µg of
pSV--gal plasmid (Promega) as a control for measuring transfection
efficiency. In cotransfection experiments, various amounts of CMV-p65
and CMV-p50 plasmid DNAs were added (indicated in figure legends)
together with reporter plasmid DNAs and carrier plasmid DNA so that the
total amount of DNA in each transfection assay remained constant.
Transfection of liver cells was carried out by the calcium phosphate
method (Graham and Van der Eb, 1973) with minor modifications as
described in Ray and Ray(1994). Monocytes used in transfection assays
were prepared from rabbit peripheral blood as described above.
Monocytes were transfected using the DEAE-dextran method (Sambrook et al., 1989). For LPS-stimulation of transfected monocytes,
cells were incubated in the presence of 10 µg/ml Escherichia
coli LPS (Sigma). As a measure of monitoring transfection
efficiency, cells were cotransfected with pSV-
galactosidase
(Promega) plasmid. Chloramphenicol acetyltransferase (CAT) was assayed
(Sambrook et al., 1989) using
-galactosidase-equivalent
amounts of cell extracts which were heated at 60 °C for 10 min to
inactivate endogenous acetylase. All transfections were repeated at
least three times.
Figure 1:
LPS stimulation of -AT
S-2 gene expression in monocytes. A, Northern analysis of the
total
-AT mRNA isolated from rabbit peripheral blood
monocytes grown in the absence (lane 1) or in the presence (lane 2) of LPS for 5 h. Total RNA samples were fractionated
in a 1% agarose gel containing 2.2 M formaldehyde according to
the method described by Ray et al.(1994) and hybridized to a
P-labeled
-AT cDNA probe. As a control,
the membrane was reprobed with an actin cDNA probe that reveals the
qualitative and quantitative estimation of the two samples. B,
result of an S1 nuclease assay (described under ``Materials and
Methods'') that determines the level of
-AT S-2
transcripts in the mRNA preparations from monocytes grown in the
absence (lane 3) or presence of LPS (lane 4). As a
negative control yeast transfer RNA (lane 1) was used. Lane 2 contains untreated
P-labeled S-2
isoform-specific antisense oligonucleotide only. The
S
-protected fragments were separated on a 10%
polyacrylamide, 8 M urea sequencing gel and visualized by
autoradiography.
Figure 2:
Physical map of the -AT
S-2 gene of rabbit. A genomic clone,
-AT12,
containing a 19-kb insert encompass the S-2 gene. Its identity as S-2
isoform-specific was determined by Southern hybridization using S-2
gene-specific probe. Location of the gene within a 10-kb region
spanning BamHI and SalI restriction sites was
determined by Southern hybridization analysis with the
-AT S-2 cDNA probe (Ray et al., 1994), and a
restriction map was generated. The relative position and size of the
exons, identified by Roman numerals, were determined by DNA
sequence analysis of the exons and the surrounding intronic regions.
Transcription start site is indicated by an arrow.
Figure 3:
Nucleotide sequence of the promoter region
of -AT S-2 gene. Analysis of the DNA sequence spanning
-2438 to +53 reveals the transcription start site,
designated by an arrow, TATA element (boxed), and
several potential structural elements for the binding of transcription
factors HNF1/LF-B1, HNF2/LF-A1, HNF3, C/EBP, Sp1, APRF, and NF-
B.
The exon 1 (47 nucleotides in length) is designated by bold
letters.
Figure 4:
Comparison of the promoter region of
rabbit -AT S-2 gene and human
-AT
gene. S-2 gene sequences from +47 to -964 were used for
comparison. Symbols:
, identical nucleotides; -, absence of
nucleotides; R, rabbit, and H, human sequence. The
position of the transcription start site is indicated by an arrow.
Figure 5:
Functional analysis of the promoter region
of -AT S-2 gene. A DNA fragment containing sequences
from a BamHI(-3551) to an ApaI (+70) of
the
-AT S-2 gene was subcloned in right orientation
into plasmid vector pBLCAT3. The resultant plasmid, pAT-3.5 CAT, was
used in transfection of liver and monocyte cells to assess the
inducibility of the
-AT S-2 gene promoter DNA. BNL
CL.2 liver and peripheral blood monocyte cells were transfected
separately with pBLCAT3 (as a control) and pAT-3.5 CAT plasmid DNA (10
µg of each) in the presence and absence of 25% conditioned medium
or LPS (10 µg/ml). Details of the transfection and CAT assay are
described under ``Materials and Methods.'' CAT activity was
measured as radioactivity (counts/min) in
[
C]chloramphenicol acetate produced by an
equivalent amount of transfected cell extracts. Transfections were
normalized to the
-galactosidase control activity. Plotted values
represent an average CAT activity relative to that of uninduced vector
plasmid pBLCAT3 from three separate transfection assays, and the range
represents the maximum values obtained in an individual
assay.
Figure 6:
Analysis of the LPS responsive promoter of
the -AT S-2 gene. Specific sequences of the
-AT S-2 gene (horizontal bars) inserted
upstream of the CAT gene of pBLCAT3 vector are aligned with a schematic
locating specific restriction enzyme sites. Peripheral blood monocytes
were transiently transfected with the constructs indicated (10 µg
of DNAs were used for each plasmid). After transfection, cells were
maintained in the medium in the absence of stimulation or treated with
10 µg/ml of LPS for an additional 24 h. The histogram represents
uninduced (solid bars) and LPS-induced (hatched bars)
CAT activity. Details of transfection and the CAT activity assay are
described under ``Materials and Methods.'' Relative CAT
activity was determined as described in Fig. 5.
Figure 7:
Distal promoter element of
-AT S-2 gene interacts with the LPS-induced nuclear
factors. EMSAs were performed with
P-labeled
-AT S-2 promoter DNA containing a NF-
B element
(-2295 to -2250). A, nuclear extracts (10 µg
of protein), prepared from normal untreated (lane 1) and 4-h
LPS-treated (lane 2) monocytes, were incubated with the
P-labeled probe following the method described under
``Materials and Methods,'' and the resulting complexes were
resolved in a 6% native polyacrylamide gel. The positions of two
DNA-protein complexes A and B formed by the factors
in the LPS-induced nuclear extract are shown. B, prior to the
addition of
P-labeled probe, the LPS-induced nuclear
extract was incubated with competitor oligonucleotides containing
either the wild-type (wt) (lane 2) or mutant (mt) (lane 3) sequence for the NF-
B element. The
sequences of these two oligonucleotides are described under
``Materials and Methods.'' A control assay containing no
competitor oligonucleotide is shown in lane
1.
Figure 8:
Identification of NF-B/Rel family
members that interact with the distal promoter element of the
-AT S-2 gene. EMSA was performed using
P-labeled promoter DNA (-2295 to -2250) and
nuclear extract (10 µg of protein) from LPS-treated rabbit blood
monocytes. Prior to the addition of the
P-labeled probe,
nuclear extract was incubated with 0.5 and 1 µl of a 10-fold
diluted anti-p50 antisera (lanes 2 and 3,
respectively) or anti-p65 antisera (lanes 4 and 5,
respectively). These antisera have been described earlier (Ray et
al., 1995). As a control, a nonspecific antisera was also used (lane 6). The preincubation was done at 4 °C for 45 min
and then incubated with the
P-labeled probe. Lane 1 contains no antisera. The DNA-protein complexes were resolved in a
6% native polyacrylamide gel.
Figure 9:
Site-directed mutagenesis of the NF-B
element abolishes LPS inducibility. Peripheral blood monocytes were
transiently transfected with 10 µg of CAT reporter plasmids. The
pAT-2.4 CAT plasmid contained wild-type S-2 promoter
(-2438/+70) DNA, and the pAT-2.4mtNF-
B CAT plasmid
contained the same DNA selectively mutated at the NF-
B-binding
element. The sequence of the wild-type (wt) and mutant (mt) NF-
B elements are indicated. The underlined
bases represent mutated bases. After transfection, one set of
transfected cells were stimulated with 10 µg/ml LPS for 24 h.
Relative CAT activity was determined as described in Fig. 5.
Figure 10:
Overexpression of NF-B potentiates
-AT S-2 promoter activity. Two CAT reporter plasmids
(10 µg each), pAT-2.4 CAT and pAT-2.4mtNF-
B CAT, carrying
either wild-type S-2 promoter DNA (-2438/+70) or the same
DNA selectively mutated at the NF-
B site, respectively, were
cotransfected in blood monocyte cells with increasing amounts (0, 1, 2,
3, 4, and 5 µg) of the plasmids expressing either NF-
B (p65)
or NF-
B (p50) proteins. Relative CAT activity was determined as
described in Fig. 5.
Extremely high inducibility of the -AT S-2
gene under inflammatory conditions has prompted a detailed analysis of
the regulatory elements of this gene.
-AT is one of
the major proteinase inhibitors in serum with a broad range of
activities but mainly protects the elastic fibers in lungs from
excessive digestion by neutrophil elastase. During the host response to
inflammation/injury expression of
-AT is induced.
However, the molecular mechanisms of this induction process is still
unclear. This report presents the first clear evidence for a regulatory
element that controls the inducible expression of an inflammation
responsive
-AT gene. The major findings of the present
study include identification of a distal LPS-responsive promoter region
that contains a NF-
B-binding element. This element binds with
NF-
B, and its mutation abolishes the LPS-mediated inducibility of
-AT promoter construct in monocytic cells.
Overexpression of NF-
B in monocytes induced the
-AT expression. The data taken together strongly
suggest that LPS-mediated
-AT gene induction is
controlled by NF-
B. Involvement of NF-
B in the
-AT gene induction process has not been reported
earlier.
What is the significance of the high level induction of the
-AT S-2 gene under inflammatory conditions? A logical
explanation may be to down-regulate the proteolytic cascade associated
with the cell death pathway activated during inflammation. Interleukin
1-
produced and activated in response to inflammation can activate
many cellular events causing cell death. IL-1
-converting enzyme
(ICE), a mammalian proteinase processes pro-IL-1
to the active
form mature IL-1
by proteolytic cleavage at aspartic residues
(Black et al., 1988). ICE is also homologous to the ced-3 gene of Caenorhabditis elegans that is essential for
apoptosis (Ellis et al., 1991). Recently it has been shown
that ICE can be inhibited by a novel cysteine proteinase inhibitor CrmA
originally identified in cowpox virus (Ray et al., 1992).
Expression of crmA can prevent cell death mediated by ICE as
well as quelling other IL-1-mediated inflammatory response.
Overexpression of crmA can also prevent cell death mediated by
TNF receptor associated death domain protein (Hsu et al.,
1995), Fas associated death domain protein (Chinnaiyan et al.,
1995), and a death protease Yama, a mammalian homolog of ced-3 (Tewari et al., 1995). In view of high inducibility of
the
-AT S-2 gene under inflammatory conditions, it is
possible that a rabbit genome has evolved and maintained this highly
inducible proteinase inhibitor to maintain cellular homeostasis and
cope with the influx of proteinases brought in by infectious agents
upon wounding or infection. It is known that parasites such as Schistosoma mansoni (McKerrow et al., 1985) and
bacteria such as Pseudomonas (Werb et al., 1982)
synthesize serine proteinases that act as virulence factors to increase
the efficiency of infection.
In an effort to elucidate the mechanism
of transcriptional induction, we undertook the cloning and
characterization, as well as sequencing, of the immediate upstream
promoter region of the rabbit -AT S-2 gene. This gene
spans about a 10-kb region and consists of five exons. A translation
initiator ATG codon is present at the beginning of the second exon,
while the first exon codes for the 5`-untranslated region. The proximal
promoter region, up to 200 bp upstream of the transcription start site (Fig. 4) of S-2 gene, shows a high degree of homology with the
proximal region of human and mouse
-AT genes. This
region contains a number of major transcription factor-binding elements
including HNF1/LF-B1, HNF2/LF-A1, HNF3, and C/EBP which are seen to be
highly conserved. Such a high conservation of several transcription
factor binding sequences argues for their importance in controlling the
basal expression of
-AT across the species. These
elements, however, are not sufficient for the inflammation-induced
expression of the S-2 gene. Additional regulatory elements are crucial
for its inducibility. This is evident from the detailed promoter
analyses of this gene (Fig. 6).
Functional studies show that
promoter of the S-2 isoform is highly inducible both in liver and
monocyte cells in response to cytokines present in the conditioned
medium or LPS (Fig. 5). Transient transfection of monocyte cells (Fig. 6) using a series of CAT gene reporter constructs,
demonstrates that upstream sequences located between -2438 and
-1990 (a 0.5-kb PstI fragment) are important for the
induction response mediated by LPS. Analysis of the LPS-responsive
region of S-2 promoter revealed the presence of a NF-B-binding
element in this region. Drastic reduction of LPS responsiveness, when
the NF-
B-binding element is altered, clearly suggests that
NF-
B plays a crucial role for efficient induction of the
-AT S-2 gene by LPS in monocytic cells. The abilities
of (i) inducible NF-
B-like factors to bind to the
-AT promoter, (ii) anti-p65 and anti-p50 antibodies to
supershift the LPS-inducible DNA-protein complex to the
-AT promoter, and (iii) the overexpression of p65 and
p50 subunits of NF-
B to activate
-AT gene
expression further attest to the role of NF-
B as a regulator of
-AT gene induction. NF-
B is a family of
pleiotropic inducible transcription factors originally identified as
nuclear factors that bind to the
B enhancer motif of
immunoglobulin
light chain (Sen and Baltimore, 1986). The
transcription factor is activated by a number of extracellular signals
and cytokines including IL-1 and tumor necrosis factor. Recent numerous
studies have shown that NF-
B regulates a wide variety of genes
including those of IL-1
, IL-6, IL-8, IL-2, and serum amyloid A
(Bensi et al., 1990; Libermann and Baltimore, 1990; Stein and
Baldwin, 1993; Hoyos et al., 1989; Ray et al., 1995).
The involvement of NF-
B in the transcriptional induction of
-AT broadens the range for this factor to yet another
gene that is induced in response to inflammatory stress.
In this
report, we have laid the foundation for more detailed analyses of the
regulation of the inducible expression of the -AT gene
in liver and monocyte/macrophages during the inflammatory response.
Increased expression of
-AT also poses a serious
health problem in individuals carrying the P
-AT mutant allele. During inflammation,
synthesis of
-AT in these patients is increased,
causing greater intracellular accumulation of the defective protein
(Barbey-Morrel et al., 1987). Understanding the molecular
mechanisms of inducible expression of
-AT will allow
for development of therapies to intervene the increased synthesis and
accumulation of the defective protein in the liver of a homozygous
P
individual. Further work along this line is
currently in progress.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L42320[GenBank].
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