From the Department of Ophthalmology and Visual
Sciences, University of Illinois at Chicago College of Medicine,
Chicago, Illinois 60612 and the § Departments of
Biochemistry and Ophthalmology, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
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ABSTRACT |
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The transcripts of the
1-proteinase inhibitor in the cornea are different
from those in hepatocytes and monocytes, suggesting that
1-proteinase inhibitor gene transcription may respond to different cell-specific regulatory mechanisms. Although information on
1-proteinase inhibitor gene structure has been obtained,
little is known regarding the cis- and
trans-acting factors that regulate its expression. In this
study, we cloned and sequenced a 2.7-kilobase 5'-flanking region
upstream from the corneal transcription initiation site of the gene,
demonstrated functional promoter activity, and identified the
regulatory elements. Sequencing revealed that the 5'-flanking element
was highly G/C-rich in regions proximal to the corneal transcription
start site. DNase I footprinting located 10 potential Sp1-binding sites
between nucleotides
1519 and +44. The putative promoter was
functional in human corneal stromal cells, but not in human skin,
scleral, and conjunctival fibroblasts, suggesting that the promoter may
be corneal cell-specific. The promoter activity in the corneal cells
was repressed when Sp1 was coexpressed. In the cornea-thinning disease
keratoconus, down-regulation of the
1-proteinase
inhibitor gene and increased Sp1 expression have both been
demonstrated. The current results suggest that down-regulation of the
inhibitor in keratoconus corneas may be related directly to
overexpression of the Sp1 gene. This information may help
elucidate the molecular pathways leading to the altered
1-proteinase inhibitor expression in keratoconus.
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INTRODUCTION |
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Proteinase inhibitors are critical in preventing and controlling
proteolysis. The 1-proteinase inhibitor
(
1-PI)1 is a
major protease inhibitor in human serum (1). One of its primary
physiologic roles is to protect the elastic fibers in lung alveoli from
excessive digestion by neutrophil elastase (2, 3). The importance of
this protein was proposed in the 1960s based on observations that
genetically
1-PI-deficient patients developed an
early-onset degenerative lung disease (4) or a liver disease (5).
The liver is the predominant site of
1-PI synthesis (1).
This protein is also found synthesized in blood monocytes and
macrophages (6), alveolar macrophages (7), intestinal epithelial cells
(8), and human breast carcinoma (9). Recently, the synthesis of
1-PI by human corneal cells has been reported (10).
The cornea, located in the anterior portion of the eye, is a
transparent connective tissue made up of epithelial, stromal, and
endothelial layers. The balance between proteinases and proteinase inhibitors is believed to play a significant role in maintaining normal
cellular contents and normal function of the cornea (10). A proper
level of 1-PI may protect the cornea from degradation by
neutrophil elastase during inflammation. Additionally,
1-PI may function as a backup inhibitor for other serine
proteinases such as plasmin and cathepsin G in the cornea.
In an ocular disease called keratoconus, 1-PI expression
is reduced to one-third to one-fifth of the normal level, and the expression of degradative enzymes is increased (11, 12). This disease
is a noninflammatory disorder that progressively thins and distorts the
central portion of the cornea and leads to visual impairment (13). The
etiology is unclear, although one hypothesis is that the abnormality in
keratoconus may lie in the degradative pathway of macromolecular
constituents in the cornea (14). A reduction in the
1-PI
level certainly would have a direct impact on the degradation
processes, contributing to keratoconus conditions.
The 1-PI gene has been localized on chromosome 14. It
contains seven exons: Ia, Ib, Ic, and II-V (15).
1-PI
transcripts that comprise different numbers of exons have been
identified. The
1-PI mRNA from the liver and
intestinal epithelium contains five exons (Ic and II-V), with a single
transcription start site in the middle of exon Ic (16). The major
promoter elements for this transcript are found in exon Ic and in the
intron between exons Ib and Ic. In blood monocytes, multiple
transcripts exist; all seven exons can be expressed, or exons Ib and Ic
can be spliced out (17).2 In
addition, three transcription initiation sites (Fig.
1) were noted: two in exon Ia and the
third in exon Ib (16). The promoter region upstream from exon Ia has
not previously been studied.
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In the cornea, alternatively spliced forms have been found by reverse
transcription-polymerase chain reaction (PCR). One of these forms is
similar in size to that found in monocytes that has exons Ib and Ic
spliced out,2 and the other involves an alternative
splicing between exons Ib and Ic. The transcription initiation site for
the cornea-specific form (Fig. 1) is ~2 kb upstream from the
hepatocyte site between the two macrophage sites in exon
Ia.2 The translation start site, ~7.4 kb downstream from
the corneal transcription initiation site (18), is the same for
hepatocytes, monocytes, and the cornea. The use of different
transcription start sites for the 1-PI gene and the
alternative splicing in different cells suggest that the gene
transcription may respond to cell-specific regulatory mechanisms
(17-20). Although liver-specific
1-PI gene regulation
has been intensively studied (19, 20), there is relatively little
information regarding the cis- and trans-acting
factors directing
1-PI gene expression in
extrahepatocytic cell types.
In this study, a 2.7-kb 5'-flanking DNA upstream from the corneal
transcription initiation site of the 1-PI gene was
sequenced; its functional activity in corneal cells was demonstrated;
and the regulatory elements of this putative promoter were
investigated. Emphasis was placed on the binding sites for
transcription factor Sp1 and gene regulation by Sp1 because the Sp1
level has been found to be increased 10-15-fold in keratoconus corneas
(21). Our results suggest that the 5'-flanking element identified may be cornea-specific, that Sp1 appears to be involved in regulation of
the promoter activity, and that the down-regulation of the
1-PI gene observed in keratoconus corneas may be
directly related to the overexpression of Sp1. This information may
help elucidate the molecular pathways leading to the altered
1-PI expression in keratoconus.
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EXPERIMENTAL PROCEDURES |
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Materials
Corneal stromal cells were cultured from normal human corneas obtained from the Illinois Eye Bank. The corneal cells, skin fibroblasts derived from skin tissues, and scleral and conjunctival fibroblasts derived from donor eyes were grown and maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with glutamine, 10% (v/v) fetal calf serum, nonessential and essential amino acids, and antibiotics as described previously (22).
For PromoterFinder experiments, a pair of sense strand PCR primers,
gene-specific for 1-PI (Gsp1 and Gsp2), were selected through the computer program Oligo Version 4.1 (National Biosciences, Plymouth, MN) from the known human exon Ia genomic DNA sequence of
1-PI (17, 19) and were used to amplify the upstream
region. Primer sequences were as follows: Gsp1, GTAGACTTCGGGTGGAGGCAGT; and Gsp2, GGGGAGCTTGGACAGGAAG. Primers were synthesized by Genemed Biotechnologies, Inc. (South San Francisco, CA).
Methods
PromoterFinder, Cloning, and Sequencing--
The PromoterFinder
DNA Walking kit (CLONTECH, Palo Alto, CA) was used
to amplify the upstream region. This kit provides five human genomic
libraries, each exhaustively digested with one of five restriction
enzymes and manipulated to have specific known sequences attached to
either end of all digested fragments. A pair of primers specific for
these attached sequences (Ap1 and Ap2) were used in conjunction with
the 1-PI gene-specific primers (Gsp1 and Gsp2) in five
long PCRs (one for each library) using the Expand Long Template PCR
system (Boehringer Mannheim). The primary round of PCR was carried out
with the outer primers (Ap1 and Gsp1) with seven cycles at 94 °C for
2 s and 72 °C for 3 min, 32 cycles at 94 °C for 2 s and
67 °C for 3 min, and 1 cycle at 67 °C for 4 min using the GeneAmp
2400 Cycler (Perkin-Elmer). One microliter of a 1:50 dilution of the
primary PCRs was subjected to a second nested long PCR using the inner
primers (Ap2 and Gsp2) with five cycles at 94 °C for 2 s and
72 °C for 3 min, 20 cycles at 94 °C for 2 s and 67 °C for
3 min, and 1 cycle at 67 °C for 4 min. The PCR products were
analyzed on a 1.2% agarose gel and cloned into the pGEM-T Easy vector
(Promega, Madison, WI). DNA sequencing was performed using the
Sequenase Version 2.0 DNA sequencing kit (U. S. Biochemical Corp.) and
the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit
(Perkin-Elmer). The sequences were analyzed by the MacVector computer
program.
Southern Blot and Restriction Digestion Analysis--
A 2712-bp
(2703 to +9)
1-PI 5'-flanking region obtained from a
long PCR was ligated into the multiple cloning sites of the pSEAP2-Basic vector. This plasmid was digested with restriction enzymes
and separated on a 1% agarose gel. Southern blotting was performed
according to the Boehringer-Mannheim nonradioactive detection protocol
using Hybond N membranes (Amersham Pharmacia Biotech). The two DNA
probes (probes 1 and 2) were selected and prepared by PCRs using
genomic DNA as the template. The DNA sequence of probe 1 (
2695 to
1977) was according to the sequence we identified, and that of probe
2 (
172 to +44) was from the sequence published in the literature
(17). Probes 1 and 2 were randomly labeled using a nonradioactive
labeling kit. Restriction digestions using endonucleases
ApaI, SmaI, and PstI were performed to
further prove the identity of the
1-PI 5'-flanking
sequence we identified.
DNase I Footprinting--
Four DNA fragments covering 1563 bp
(1519 to +44) of the
1-PI promoter region were
prepared from genomic DNA by PCR (fragment 1,
338 to +44; fragment 2,
782 to
321; fragment 3,
1200 to
731; and fragment 4,
1519 to
1115). They were 5'-end-labeled with [
-32P]ATP using
T4 polynucleotide kinase. Footprinting was performed using the labeled
DNA fragments (30 ng) and human Sp1 transcription factor from Promega
(4 or 2 footprinting units) according to the Promega core footprinting
protocol. In the competitive footprinting assay, 50- and 100-fold molar
excesses of unlabeled Sp1 oligonucleotide competitors (Promega) were
included in the reaction mixture. Purine sequence ladders of DNA probes
were prepared (23) and subjected to gel electrophoresis on urea
sequencing gels adjacent to footprint reactions of the same probe to
localize binding site sequences.
Promoter Activity Analysis--
The series of secreted alkaline
phosphatase (SEAP) vectors available from CLONTECH
were used for promoter-reporter plasmid constructs. A 2712-bp (2703
to +9)
1-PI promoter fragment and a 1406-bp (
1397 to
+9) fragment obtained from a long PCR were ligated into the
KpnI and BglII multiple cloning sites of the pSEAP2-Basic vector, yielding the p2.7SEAP+ and
p1.4SEAP+ vectors. The plasmids p2.7SEAP+,
p1.4SEAP+, pSEAP2-Basic, and pSEAP2-Control (positive
control, driven by the SV40 early promoter) and pSV-
gal (Promega)
used in cell transfections were purified by QIAGEN ion-exchange columns
and were partially sequenced or restriction-digested to confirm their
identity and orientation. Human corneal stromal cells and skin,
scleral, and conjunctival fibroblasts were plated at 0.9 × 106 cells/well on six-well plates 24 h before DNA
transfection. Three hours before transfection, the dishes received
fresh medium. Cells were transfected by the calcium phosphate method
using the CalPhos Maximizer transfection kit from
CLONTECH. In brief, 20 µg of the test plasmid
(p2.7SEAP+, p1.4SEAP+, pSEAP2-Basic, or
pSEAP2-Control) along with 2 µg of pSV-
gal vector, used to control
transfection efficiency, were mixed with 40 µl of CalPhos Maximizer
as recommended by the manufacturer. Some cells also received 2 or 4 µg of Sp1 expression vector pPacSp1. None of the test plasmids were
added to cells serving as negative controls. After incubation with the
DNA mixture for 4 h, fresh culture medium was added to the cells,
and the medium was collected 72 h later for SEAP assay. Cells that
received no DNA were used as negative controls. For SEAP activity, 15 µl of cell medium was mixed with 45 µl of 1× dilution buffer and
60 µl of assay buffer according to the manufacturer's protocol
(CLONTECH). For the enzyme activity, the absorbance
was read for 1 s on a Luminometer (Wallac, Gaithersburg, MD). For
-galactosidase assays, cells were harvested, washed in
phosphate-buffered saline, resuspended in 150 µl of 0.25 M Tris (pH 8.0), lysed by freezing and thawing, and
centrifuged at 14,000 × g for 20 min at 4 °C. The
extract was mixed with Galacto-Lysis solution (Tropix Inc., Bedford,
MA) as recommended by the manufacturer. Ten microliters of lysate were
used for the Bradford protein assay (24). The
-galactosidase activity was used to normalize the SEAP enzyme activity. Assays were
performed in triplicate, and each experiment was repeated at least
three times. Two-tailed Student's t tests were used to analyze the significance of the data.
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RESULTS |
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Isolation and Sequencing of the Corneal 1-PI
5'-Flanking Element--
The corneal 5'-flanking region of the human
1-PI gene was isolated using the PromoterFinder kit
(Fig. 2). Three products (2.6, 2.0, and
0.8 kb) were obtained from the EcoRV library. Two PCR
fragments (0.6 and 0.28 kb) were obtained from the ScaI and PvuII libraries, respectively. From the SspI
library, three PCR fragments (1.3, 1.0, and 0.7 kb) resulted. No
product was observed in the DraI library.
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Identification of the Sp1-binding Sites--
Sp1 binding to the
5'-flanking element of the 1-PI gene was further
examined by DNase I footprint analysis. Ten regions in the 1563-bp
(
1519 to +44) portion of the 5'-flanking DNA were found to be
protected by Sp1 against DNase I digestion (Fig.
5, A-D); regions 1-10
covered from nucleotides
100 to
87;
301 to
290,
409 to
403,
519 to
498,
593 to
579,
622 to
612,
672 to
666,
819
to
793,
932 to
915, and
998 to
987, respectively. The 10 binding sites are marked by asterisks in the sequence shown in Fig. 3. Five of the regions (boxed and marked by
asterisks) correlated with the Sp1-binding sites identified
by MacVector. The remaining five (marked by asterisks) were
G/C-rich regions. The Sp1 binding was specific because the degree of
DNase I protection was reduced in the presence of a competing Sp1
oligonucleotide.
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Functional Analysis of the Corneal 1-PI 5'-Flanking
Element--
The activity of this putative
1-PI gene
promoter in normal human corneal stromal cells and in skin, scleral,
and conjunctival fibroblasts was investigated in transient transfection
assays. For corneal stromal cells, DNA fragments containing 1406 bp
(p1.4SEAP+) and 2712 bp (p2.7SEAP+) of the
5'-flanking sequence were approximately six times more active at
driving SEAP reporter gene expression than the pSEAP2-Basic vector
(Fig. 6). No such activity was found,
indicating that neither segment of the 5'-flanking DNA was functional
in skin, scleral, and conjunctival fibroblasts. When corneal stromal
cells were cotransfected with pPacSp1, the level of SEAP expression was
markedly reduced (Fig. 7).
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DISCUSSION |
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This study provides the first comprehensive structural and
functional analysis of the 5'-flanking element of the human
1-PI gene upstream from the corneal transcription start
site. The 2.7-kb region sequenced is characterized by multiple binding
sites for transcription factor Sp1 and the absence of a consensus TATA
box. Transient transfection experiments showed that the 2.7-kb
5'-flanking DNA is functional in human corneal stromal cells and that
the proximal 1400 base pairs are sufficient for full promoter activity. No promoter activity was found for either the 2.7- or 1.4-kb segment in
human skin, scleral, and conjunctival fibroblasts, suggesting that the
5'-flanking element we identified may be specific for corneal cells.
Consistent with this notion, the cornea-specific alternatively spliced
form of
1-PI was not found in the skin dermis or the
sclera, although the form similar to that identified in monocytes was
expressed (data not shown).
Multiple start sites of transcription by primer extension have been
found in the human 1-PI gene (25). The
1-PI gene appears to belong to a class of eukaryotic
genes (namely, housekeeping genes) that are constitutively expressed at
a basal level. The promoters of these genes are characterized by a high
G/C content, by multiple binding sites for Sp1, by the absence of a
consensus TATA box, and by the feature that they initiate transcription from multiple sites spread over a fairly large region (26).
Several studies have shown that alternative promoters are used for
different 1-PI transcripts in hepatocytes and
macrophages (17, 27). For hepatocytes, the minimal promoter element
required for liver-specific basal expression of human
1-PI is confined within 261 nucleotides from the
transcription start site (19, 20). Binding sites for transcription
factors such as LF-A1/HNF2 (28), LF-B1/HNF1 (29, 30),
CCAAT/enhancer-binding protein (20, 30, 31), HNF3 (32), and Sp1 (33)
have been noted within this segment, and the first two factors were
found to be essential for liver-specific expression of
1-PI (20, 34). LF-A1 and LF-B1 also bind to a series of
other liver-specific genes, positively regulating their expression in
the liver (20, 35). Sequence analysis of the corneal
1-PI 5'-flanking element using the MacVector computer
program and/or DNase footprinting experiments demonstrated numerous
binding sites for transcription factors, including Sp1, AP-1, and
NF-
B, but no LF-A1 or LF-B1 sites were identified. Also, no homology
was found between the liver and the putative corneal promoters. This
suggests that the specificity and efficiency of gene transcription may
depend on cell-specific transcription factors. Complex interactions
with other factors may also be involved.
The regulation of 1-PI expression in the cornea has not
been previously investigated. The current results show that the
1-PI promoter activity in corneal cells was repressed by
overexpression of Sp1. This indicates that the Sp1 sites are involved
in the regulation of the
1-PI gene. In this regard, it
is of interest to note that in keratoconus corneas, in which gene
expression of
1-PI is reduced, Sp1 expression is found
increased in both epithelial and stromal layers. Sp1 binding activity
has also been shown to be markedly enhanced in nuclear extracts from
the epithelium of keratoconus corneas. The expression of four other
transcription factors studied, AP-1, AP-2, cAMP-responsive
element-binding protein, and NF-
B, remains unaltered (21). The Sp1
abnormality is also cornea-specific and is not found in either the
conjunctiva or the skin.3
Sp1 is a specific factor originally described as required for SV40 transcription. It interacts with GC boxes in the promoter elements and plays an important role in the expression of many viral and cellular genes (36-38). Recent investigations have shown that the activity and synthesis of Sp1 are subject to a variety of regulations. For example, Sp1 expression is increased during SV40 infection of CV1 cells (39). Although ubiquitously expressed, the level of Sp1 protein expression varies widely among different cell types in the mouse, and increased Sp1 expression has been associated with late stages of differentiation (38). Elevated levels of Sp1 expression have also been noted in gastric carcinoma cells (40), but keratoconus is the only human disease known to exhibit altered Sp1 expression.
Our demonstration that the 1-PI promoter activity is
suppressed by expression of Sp1 in corneal cells suggests that Sp1 may play a significant role in the regulation of the
1-PI
gene in the cornea during development, normal homeostasis, or under
pathologic conditions. In the cornea-thinning disease keratoconus, the
reduction of the
1-PI level may be directly related to
increased Sp1 expression.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert Tjian for providing plasmid pPacSp1, R. Brent Whitelock for performing pilot experiments, Drs. John Varga and Shu-Jen Chen for invaluable suggestions during the course of this study, and Dr. Theodore Perl for supplying keratoconus tissues.
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FOOTNOTES |
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* This work was supported in part by Grants EY03890 and EY05628 (to B. Y. J. T. Y.) and EY06663 (to S. S. T.) and Core Grant EY01792 from the National Eye Institute, National Institutes of Health (Bethesda, MD).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number 88408.
¶ Recipient of a senior scientific investigator award from Research to Prevent Blindness, Inc. (New York). To whom correspondence and reprint requests should be addressed. Tel.: 312-996-6125; Fax: 312-996-7773; E-mail: u24184{at}uic.edu.
1
The abbreviations used are: 1-PI,
1-proteinase inhibitor; PCR, polymerase chain reaction;
kb, kilobase(s); bp, base pair(s); SEAP, secreted alkaline
phosphatase.
2
G. Bokovic and S. S. Twining,
submitted for publication.
3 L. Zhou, I. Maruyama, Y. Li, J. Sugar, and B. Y. J. T. Yue, unpublished results.
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REFERENCES |
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