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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1256-1262
Expression of Bomapin, a Novel Human Serpin, in Normal/Malignant
Hematopoiesis and in the Monocytic Cell Lines THP-1 and AML-193
By
Matthias Riewald,
Trinette Chuang,
Andreas Neubauer,
Hanno Riess, and
Raymond R. Schleef
From the Department of Vascular Biology, The Scripps Research
Institute, La Jolla, CA and Virchow Klinikum, Department of Hematology
and Oncology, Humboldt Universität, Berlin, Germany.
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ABSTRACT |
Our group recently cloned the cDNA-encoding bomapin, a member of the
serine protease inhibitor (serpin) superfamily, from a human bone
marrow cDNA library (J Biol Chem 270:2675, 1995). To
understand its expression within the hematopoietic compartment, RNA
extracted from bone marrow or peripheral blood from normal donors and
patients with leukemia was reverse transcribed and analyzed by
polymerase chain reaction (PCR). Bomapin PCR products were readily
detected in normal bone marrow, which was designated as a medium mRNA
level. In peripheral blood, bomapin expression was low or undetectable
in normal donors (n = 6) and patients with chronic lymphocytic
leukemia (n = 6). Blood from patients with chronic myeloid leukemia
(n = 6), chronic myelomonocytic leukemia (n = 6), acute myeloid
leukemia (n = 5), and acute lymphocytic leukemia (n = 5) exhibited
low to medium levels of bomapin expression. Furthermore, a high level
of bomapin expression was detected in one individual with acute
monocytic leukemia. These data suggest that bomapin expression may be
elevated in hematopoietic cells of monocytic lineage. Therefore, we
analyzed the expression of bomapin within cell lines that exhibited
characteristics of the monocytic lineage. Bomapin PCR products were
detected in the monocytic THP-1 and AML-193 cell lines but not in CRL
7607, CRL 7541, KG-1, or K562 cells. Induction of bomapin transcripts
was not detected in the latter series of cell lines following a 24-hour
treatment with phorbol myristate acetate (PMA, 10 8
mol/L) or tumor necrosis factor- (TNF-, 30 U/mL), whereas
treatment of THP-1 or AML-193 cells with these agents reduced the
intensity of the bomapin PCR products. Northern blotting confirmed
these results and showed that the expression of bomapin in THP-1 cells was downregulated over a 4-day period by PMA and, to a lesser extent,
TNF-. Immunoblotting was used to show the presence of a 40-kD
protein in THP-1 cytosol preparations. Bomapin antigen levels were
correspondingly reduced after treatment with PMA. Because PMA and
TNF- induce monocytic differentiation in THP-1 and AML-193 cells,
these data increase the possibility that bomapin may play a role in the
regulation of protease activities specifically in early stages of
cellular differentiation.
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INTRODUCTION |
SERINE PROTEINASE inhibitors (serpins)
are a large superfamily of homologous proteins that resemble
1-proteinase inhibitor in overall structure and form
stoichiometric 1:1 inhibitory complexes with target proteases that are
typically stable to treatment with denaturants (eg, sodium dodecyl
sulfate; SDS).1,2 Serpins play crucial roles in the
neutralization of extracellular serine protease activities that are
involved in a wide variety of vital processes including blood
coagulation, fibrinolysis, complement activation, inflammation, and
cell migration.2 Well-characterized examples are the
inhibition of thrombin by antithrombin III, tissue-type plasminogen
activator by plasminogen activator inhibitor-1, or elastase by
1-proteinase inhibitor.2 Within the serpin
superfamily, ovalbumin represents the parent prototype of a currently
emerging family of structurally related proteins
(ov-serpins).3 Human members of the ov-serpin family are
plasminogen activator inhibitor-2 (PAI-2),4 an elastase
inhibitor isolated from monocyte-like cells,5
squamous cell carcinoma antigen,6 cytoplasmic
antiproteinase (CAP),7,8 and a tumor suppressor
called maspin.9 In addition, two serpins related to CAP
have been cloned from a placental  gtII library (ie, protease
inhibitor-8 and -9),10 and recent data suggest that the
latter molecule is an intracellular granzyme B inhibitor that is
associated with cytotoxic lymphocytes.11
During studies investigating the presence of protease inhibitors in
hematopoiesis, our group used a polymerase chain reaction (PCR)-based
homology cloning strategy to identify a novel member of the ovalbumin
family of serpins, which exhibited a high amino acid homology (48%)
with PAI-2, CAP, and human leukocyte elastase inhibitor.12
The isolated cDNA contains a single large open reading frame that
encodes a 397-amino acid protein.12 Northern blotting
analysis with this cDNA showed a single 2.3-kb bomapin transcript that
was expressed in human bone marrow cells but was undetectable in all
other analyzed human tissues, and this molecule was designated bone
marrow-associated serpin (bomapin). Support for a role of this molecule
in the inactivation of proteases initially was derived by the formation
of SDS-stable complexes between either thrombin or trypsin and
35S-methionine-labeled bomapin produced by an in vitro
transcription/translation system.12 In this study, we
extend our initial observations by analyzing transcription patterns for
bomapin in bone marrow and peripheral blood of normal volunteers and
patients with various forms of leukemia. Our ability to detect bomapin
transcripts at elevated levels in patients with certain types of
myeloid leukemia led us to expand our analysis to include a series of
cell lines that exhibited defined characteristics of the monocytic
lineage. Data are presented indicating that bomapin is constitutively
expressed in THP-1 and AML-193 cells, and that treatment with agents
that induce monocytic differentiation (eg, phorbol ester) cause a
downregulation of bomapin mRNA and antigen, thus raising the
possibility that bomapin plays a role in the regulation of protease
activities specifically in early stages of cellular differentiation.
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MATERIALS AND METHODS |
Bone marrow and blood samples.
Normal donors and patients were from Virchow Klinikum, Berlin, Germany,
and diagnosis was made according to standard clinical and laboratory
criteria.13 Bone marrow samples (3 mL, 20 U heparin/mL) from 6 normal bone marrow donors before allogeneic bone marrow transplantation and 7 patients with acute myeloid leukemia
(AML; median blast count 96%, range, 80% to 100%) were aspirated
after informed consent. Peripheral blood (10 to 20 mL, 20 U heparin/mL) was drawn from 6 normal donors and 29 patients with leukemia. In AML
and acute lymphocytic leukemia (ALL), the median percentage of blast
cells was 89% (range, 50% to 100%). In chronic lymphocytic leukemia
(CLL), chronic myeloid leukemia (CML), and chronic myelomonocytic leukemia (CMML), the median leukocyte count was 87 × 109/L (range, 43 to 179 × 109/L). To
isolate leukocytes for RNA extraction, 10 mL heparinized blood were
mixed with 2 mL of 5% dextran in 0.9% NaCl solution. After
sedimentation of the dextran-loaded red blood cells (23°C, 40 minutes), the leukocyte-containing supernatant was aspirated, and
leukocytes were pelleted (200g, 10 minutes). To lyse remaining erythrocytes, the pellet was resuspended in 10 mL of lysis buffer (150 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA)
and incubated for 5 minutes (23°C). The leukocytes were pelleted
again (200g, 10 minutes) followed by one washing step in 10 mL
phosphate-buffered saline (PBS). Bone marrow was directly diluted 1:10
in lysis buffer (23°C, 5 minutes) and the leukocytes were isolated
and washed as described above.
Cell culture and treatment.
Two mixed cell populations derived from human bone marrow were obtained
from the American Type Culture Collection (Rockville, MD): CRL7607 was
derived from the bone marrow of a normal donor and proliferated
primarily as attached cells exhibiting a fibroblastic appearance,
whereas CRL 7541 was derived from an abnormal bone marrow sample and
exhibited a mixed morphology composed of loosely-attached, fibroblastic, and macrophage-like cells. Four defined cell lines that
proliferated in suspension were obtained from the American Type Culture
Collection: (1) K562 cells, which were derived from the blast cells of
a 53-year-old patient with CML and have been classified as a human
erythroleukemia line; (2) KG-1 cells, which were derived from the bone
marrow of a 59-year-old male with erythroleukemia that evolved into
undifferentiated acute myelogenous leukemia; (3) THP-1 cells, which
were derived from the peripheral blood of a 1-year-old male with acute
monocytic leukemia; and (4) AML-193 cells, which were established from
the blast cells of a 13-year-old female with childhood leukemia
classified as M5 acute monocytic leukemia. CRL7607 and 7541 cells were
cultured in 10% fetal bovine serum (FBS)-containing Dulbecco's
modified Eagle's medium, K562 cells were cultured in 10%
FBS-containing RPMI, KG-1 cells were cultured in 20% FBS-containing
Iscove's modified Dulbecco's medium (IMDM), THP-1 cells were cultured
in 10% FBS-containing RPMI 1640 supplemented with 0.00004%
-mercaptoethanol, and AML-193 cells were cultured in 10%
FBS-containing IMDM supplemented with 5 µg/mL transferrin (Sigma, St
Louis, MO), 5 µg/mL insulin (Sigma), and 5 ng/mL
granulocyte-macrophage colony-stimulating factor (Genzyme, Boston, MA).
RNA isolation, cDNA synthesis, and PCR.
RNA was extracted and reverse transcribed using previously described
protocols.14 PCR was performed by modifying the previously described protocols as follows: cycle 1, 94°C for 5 minutes,
55°C for 1 minute, and 72°C for 1 minute; cycles 2 to 4, 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute; cycles 5 to 31 for bomapin, CAP, and PAI-2 and cycles 5 to 23 for two control proteins (ie,  -actin and glyceraldehyde
3 -phosphate dehydrogenase [GAPDH]), 94°C for 1 minute,
55°C for 1 minute, and 72°C for 2 minutes; cycle 32 for the
three ov-serpins and cycle 24 for the two control proteins, 94°C
for 1 minute, 55°C for 1 minute, and 72°C for 10 minutes. PCR
products (10 µL) were subjected to electrophoresis on agarose gels
and visualized by ethidium bromide staining. Based on the intensities
of the bomapin bands, expression levels were classified as high,
medium, low, or undetectable (Results and Fig 1). PCR conditions were calibrated in
preliminary experiments, in which a linear relationship between cycle
number and band intensity was observed using 32 cycles for the
amplification of bomapin, CAP, and PAI-2 transcripts and 24 cycles for
transcripts encoding either -actin or GAPDH. Two sets of primers
were synthesized for bomapin based on its published
sequence12: primer set B1, CAGTGGGCCTTCAACTCTAC
(sense, base position 707 to 726) and AATTCAATGGATGGGACTCT (antisense,
base position 1,109 to 1,090); primer set B2,
ATGGGACTCTCTAGCAACATCAATCAACCAG (sense, base position 1 to 30) and
TTAGGGGGAGCATAATCTTCCAT (antisense, base position 1,195 to 1,173).
Primers for two other serpins (ie, CAP and PAI-2) and control proteins
(ie, -actin and GAPDH) were based on published nucleotide sequences:
(1) primer set C1 for CAP, TGGTTCTGGTGAATGCTGTC (sense, base position
482 to 501) and AGGTTGCGCAGGACACTCTC (antisense, base position 866 to
847)7; (2) primer set C2 for CAP, TGCTTAGGGTCGCCAACAGG
(sense, base position 260 to 279) and AGGTTGCGCAGGACACTCTC (antisense,
base position 866 to 849)7; (3) primer set for PAI-2,
ATGCAGCAGATCCAGAAG (sense, base position 244 to 261) and
TCTCCCTGTCATAACACC (antisense, base position 1,140 to
1,123)4; (4) primer set for -actin,
CCTTCCTGGGCATGGAGTCCT (sense, base position 835 to 855) and
GCACGAAGGCTCATCATTCA (antisense, base position 1,630 to
1,611)15; and (5) primer set for GAPDH,
GGTGAAGGTCGGAGTCAACG (sense, base position 42 to 61) and
ACACGGAAGGCCATGCCAGT (antisense, base position 737 to
718).16 Controls in the PCR included (1) pBluescript vectors containing the cDNA encoding either bomapin, PAI-2, or CAP (10 ng plasmid/reaction, respectively) to show specificity of the primer
pairs, (2) the vector pGAD-9 containing normal human bone marrow cDNA
library (Clontech, San Diego, CA) as a positive control (10 ng/reaction),12 (3) no cDNA added to the PCR as a negative
control, and (4) a nonreverse transcribed RNA sample as a control for
amplification of genomic DNA.
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| Fig 1.
Expression of bomapin, CAP, and -actin in normal
hematopoiesis and in hematological malignancies. RNA was isolated from
normal bone marrow (bm; lanes 1-2) and from peripheral blood of normal donors (pb; lanes 3-4) and patients with CLL (lanes 5-6), CML (lanes
7-8), CMML (lanes 9-10), AML (lanes 11-14), and ALL (lanes 15-16).
After reverse transcription, bomapin (A) and CAP (B) cDNAs were
amplified by PCR for 32 cycles using primer pairs B1 (amplification product, 403 base pairs) and C1 (amplification product, 385 base pairs), respectively. The -actin (C) cDNA was amplified for 24 cycles (amplification product, 796 base pairs). PCR products (5 µL)
were visualized by electrophoresis in a 1.5% agarose gel followed by
staining with ethidium bromide. A 1-kb DNA ladder was loaded in the
left and right lanes. Bomapin expression was scored to be low or absent
in lanes 3-6, 8, 11, and 15-16; medium in lanes 1-2, 7, 9-10, and
12-13; and high in lane 14.
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Northern blotting.
Denaturing electrophoresis of RNA in formaldehyde-containing 1%
agarose gels and transferred to Hybond-N nylon membranes (Amersham Corp, Arlington Heights, IL) was performed as described
previously.12,17 The cDNA sequence encoding bomapin was
amplified by PCR using primer set B2 in the presence of
[32P]dCTP and hybridized to the nylon membranes using
conditions previously described.12,17 A digoxigenin-labeled
human GAPDH probe was prepared by PCR using the aforementioned GAPDH
primer set to amplify a 695-bp region of the GAPDH cDNA in the presence of PCR digoxigenin labeling mixture (Boehringer Mannheim, Mannheim, Germany) and used this probe to confirm equal loading as
described.18
Purification of bomapin, antibodies to bomapin, and
immunoprecipitation/immunoblotting protocols.
The expression, purification, and characterization of bomapin will be
described in detail elsewhere (M. Riewald and R.R. Schleef, manuscript
in preparation). For the preparation of recombinant bomapin, the coding region of bomapin was excised from
pBluescript-bomapin12 using BamHI and Xho I
and ligated in frame to the cDNA-encoding glutathione S-transferase
(GST) in the bacterial expression vector pGEX-4T-1 (Pharmacia Biotech
Inc, Piscataway, NJ). PGEX-4T-1 bomapin-transformed Escherichia coli cells were induced with 100 µmol/L
isopropylthio--D-galactoside, sonicated, and centrifuged and the
supernatant was mixed with glutathione-Sepharose 4B as described by the
manufacturer. The beads were washed with (PBS) containing 1% Triton
X-100, and the GST-bomapin was eluted with PBS supplemented with 20 mmol/L glutathione and 0.1% Triton X-100. The eluted material was
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by silver staining.19,20 Fractions
containing a single protein band at 62 kD were pooled and used to raise
antibodies in New Zealand white rabbits according to standard
protocols.20 Native bomapin was isolated from THP-1 cells
using immunoblotting with antibodies to bomapin to optimize a
purification protocol. Briefly, 109 cells were homogenized
and subjected to ultracentrifugation (100,000g for 1 hour), and
the cytosol was chromatographed in a heparin-Sepharose (1 × 15 cm) column in 20 mmol/L HEPES-HCl, pH 7.4. The nonabsorbed material was
applied to a Diethylaminoethyl-Sephacryl (Pharmacia) column (1 × 15 cm) in 20 mmol/L HEPES-HCl, pH 7.4. The
column was washed and eluted with a linear gradient of NaCl (0 to 0.15 mol/L NaCl in 20 mmol/L HEPES-HCl). Fractions containing bomapin were
pooled and applied to a hydroxylapatite column in 20 mmol/L HEPES-HCl,
pH 7.4. The column was eluted by increasing the phosphate concentration
(300 mL linear gradient, 0 to 0.15 mol/L phosphate buffer, pH 7).
Fractions from containing bomapin were subjected to preparative
SDS-PAGE as a means to isolate the Mr 40 kD form of bomapin. Purified
bomapin was coupled with cyanogen bromide-Sepharose (Pharmacia) and used to affinity purify antibodies to bomapin using
previously described protocols.20 Antibodies were
biotinylated using Ezlink Sulfo-NHS-LC-Biotin (Pierce) as described by
the manufacturer. Immunoprecipitation21 and
immunoblotting20,22 procedures were performed as described
previously.
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RESULTS |
Expression of bomapin mRNA in normal and malignant hematopoietic cells.
To understand the expression of bomapin within hematopoietic tissues,
we analyzed transcription patterns in bone marrow or peripheral blood
from normal donors and patients with leukemia (Fig 1,
Table 1). Using a calibrated PCR protocol,
bomapin transcripts were readily detected in normal bone marrow, which
was designated as a medium expression level (Fig 1A, lanes 1 to 2). In
peripheral blood, bomapin expression was low or undetectable in normal
donors and patients with CLL (Fig 1A, lanes 3 to 6, Table 1). Blood from patients with CML, CMML, AML, and ALL exhibited either low or
medium levels of bomapin expression (Fig 1A, lanes 7 to 13 and 15 to
16, Table 1). However, the bomapin PCR product level was high in one
individual with acute monocytic leukemia (AML M5b; Fig 1A, lane 14). A
trend toward elevated bomapin expression was also detected in patients
with CMML in comparison with those individuals with CML (Table 1). To
substantiate our observations on bomapin, we used a second set of
primers for this molecule, which resulted in similar observations, and
representative data obtained using this set of primers will be provided
in the next section. These data suggest that bomapin expression may be
elevated in hematopoietic cells of monocytic lineage.
In comparison to the elevated expression of bomapin in individuals with
specific types of leukemias, the expression of another ov-serpin (ie,
CAP) was detected in samples of normal bone marrow and peripheral
blood, as well as in all individuals analyzed in this study. However,
one individual with acute monocytic leukemia (Fig 1A, lane 14), who
exhibited elevated expression of bomapin, appeared to contain reduced
levels of CAP expression (Fig 1B, lane 14). The PCR products for
-actin are shown in Fig 1C as a control.
Detection of bomapin in cell lines derived from patients with various
types of leukemia.
Because studies on the role of bomapin would be facilitated by the
identification of bomapin-producing cell lines that could be readily
cultured on a large scale in vitro, we investigated a series of cells
that were derived from bone marrow or peripheral blood for the presence
of bomapin transcripts by PCR. Initial experiments investigated two
mixed cell populations prepared by the Naval Biosciences
Laboratory (Oakland, CA): one derived from normal bone
marrow (CRL 7607; Fig 2, lanes 1 to 3) and
the other that was derived from an abnormal bone marrow sample (CRL
7541; Fig 2, lanes 4 to 6). Total RNA was extracted and analyzed from not only nonstimulated cells but also cells incubated for 24 hours in
the presence of phorbol myristate acetate (PMA) or tumor necrosis factor- (TNF-), two known inducers of cellular differentiation that have also been observed to increase the production of the serpins
PAI-1 and PAI-2. Although transcripts for CAP could be detected in both
cell lines (Fig 2, second row, lanes 1 to 6) and the intensity of the
PCR products for PAI-2 increased using RNA extracted from PMA (ie, CRL
7607 and 7541; Fig 2, third row, lanes 2 and 5, respectively) or
TNF- (ie, CRL 7607, lane 3) treated cells, bomapin transcripts were
not detected in these two cell populations either under nonstimulated
or stimulated conditions (Fig 2, first row, lanes 1 to 6). Our
observation that bomapin expression was elevated in certain patients
with leukemia (Fig 1, Table 1) subsequently led us to analyze the
following set of cell lines: KG-1, K562, THP-1, and AML-193. Figure 2
(first row) indicates that bomapin transcripts were present in two cell lines derived from patients with acute monocytic leukemia under normal
tissue culture conditions (ie, control THP-1 cells, lane 13; AML-193
cells, lane 16), but not in KG-1 (lanes 7 to 9) or K562 (lanes 10 to
12) cells. Both PMA and TNF appeared to cause a decrease of the
intensity of the bomapin PCR product band obtained using RNA extracted
from agonist-treated THP-1 (Fig 2, first row, lanes 14 and 15 in
comparison with lane 13) and AML-193 (Fig 2, first row, lanes 17 and 18 in comparison with lane 16) cells.
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| Fig 2.
PCR analysis of cell lines derived from bone marrow or
peripheral blood of patients with various types of leukemia. The
indicated cell lines (CRL 7607, lanes 1-3; CRL 7541, lanes 4-6; KG-1,
lanes 7-9; K562, lanes 10-12; THP-1, lanes 13-15; and AML-193, lanes 16-17) were incubated in serum-containing media (106
cells/mL, 25 mL/162 cm2 flask) in the absence (lanes 1, 4, 7, 10, 13, and 16) or presence of either PMA (10-8 mol/L,
lanes 2, 5, 8, 11, 14, and 17) or TNF- (30 U/mL; lanes 3, 6, 9, 12, 15, and 18). After 24 hours, the cells were washed by centrifugation
and total RNA was isolated, reverse transcribed, and subjected to PCR
amplification using primers specific to bomapin (first row, primer pair
B2, amplification product: 1,195 base pairs), CAP (second row, primer
pair C2, amplification product: 607 base pairs), PAI-2 (third row,
amplification product: 897 base pairs), and GAPDH (fourth row,
amplification product: 695 base pairs). The PCR products were subjected
to electrophoresis in a 1% agarose gel and stained with ethidium
bromide. Lanes 19-22 show the PCR products obtained using the
aforementioned primers to amplify the vectors containing the cDNAs
encoding bomapin (lane 19), CAP (lane 20), PAI-2 (lane 21), or GAPDH
(lane 22). Lane 23 contains a 1-kb DNA ladder.
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The levels of CAP and PAI-2 expression were also analyzed in these four
leukemic cell lines for comparative purposes. Figure 2B and C indicate
that the expression of these two serpins and their levels in response
to PMA or TNF- appear to depend on the cell line. For example, PCR
products for CAP were readily detected using mRNA from the KG-1 or K562
cell line (Fig 2B, lanes 7 and 10, respectively) and the mRNA levels
for this protein were not affected by treatment of the cells with
either agonist (Fig 2B, lanes 8, 9, 11, and 12). In comparison, CAP
transcript levels appeared to be reduced by treating the AML-193 cell
line with PMA (Fig 2B, lane 17 v lane 16), whereas the PCR
products for CAP were barely detected using mRNA from either control or
stimulated THP-1 cells (lanes 13 to 15). PCR products for PAI-2 (Fig
2C) were only detected in the K562 and AML-193 cell line after
stimulation with PMA (lanes 11 and 17, respectively), but not in mRNA
samples prepared from KG-1 or THP-1 cells (lanes 7 to 9 and 13 to 15, respectively) under control or stimulated conditions. PCR products for
a control protein (ie, GAPDH) are shown in Fig 2D.
Bomapin expression is downregulated on differentiation of THP-1 and
AML-193 cells.
To confirm that PMA and TNF- are able to decrease the steady-state
levels for bomapin mRNA, we selected THP-1 cells for further analysis.
Because cellular differentiation of THP-1 cells elicited by PMA
treatment occurs over several days, we expanded our analysis by
stimulating THP-1 cells not only for 24 hours but also over a 96-hour
period with these agonists. Total RNA was extracted from these cells
and the samples were subjected to Northern blotting using the cDNA for
bomapin-labeled with 32P. A single transcript was detected
in the RNA extracted from control THP-1 cells that were harvested
either 24 or 96 hours after their transfer into fresh media
(Fig 3, lanes 1 and 4). Although
nonstimulated THP-1 cells proliferate in suspension, their treatment
with 10-8 mol/L PMA mediates the attachment of these cells
to the culture dish (ie, >95% within 24 hours) resulting in
cessation of cell proliferation. In comparison, only a portion of the
TNF--treated cells adhered to the plastic (ie, 15% after 24 hours
decreasing to 10% at the 96-hour time point), which was accompanied by
a 23% decrease in cell growth over the 96-hour period relative to the
growth of control THP-1 cells (data not shown). Treatment of THP-1
cells with PMA or TNF- (lanes 2 and 3, respectively) for 24 hours
reduced bomapin mRNA levels below the sensitivity of this assay. The
bomapin mRNA levels in the cells treated for 96 hours with PMA remained
below the level of sensitivity of Northern blotting (lane 5), whereas
mRNA for this inhibitor could be detected again by Northern blotting in
these cells incubated for 96 hours with TNF- (lane 6).
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| Fig 3.
Detection of bomapin transcript in THP-1 cells. THP-1
cells were incubated in the absence (lanes 1 and 4) or presence of PMA (10-8 mol/L, lanes 2 and 5) or TNF- (30 U/mL, lanes 3 and 6). RNA was isolated after either 24 hours (lanes 1-3) or 96 hours
(lanes 4-6) of treatment and the blot was hybridized (10 µg/lane) to a 32P-labeled bomapin cDNA probe and exposed for 1 day to
radiograph film (A). The blot was rehybridized to a digoxigenin-labeled
GAPDH cDNA probe (25 mg/mL) and the bound probe detected by incubation with alkaline phosphatase-labeled antidigoxigenin followed by CDP-Star
(Boehringer Mannheim), and exposure to radiograph film for 10 minutes
(B).
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To investigate the production of this inhibitor on the protein level,
the cDNA encoding bomapin was subcloned in-frame after the sequence
coding for GST in pGEX-4T-1. The GST-bomapin fusion construct was
purified from bacteria transformed with this plasmid and the purified
construct was used to immunize a rabbit. Antiserum obtained from this
rabbit was affinity purified using Sepharose conjugated to purified
bomapin, and this reagent was subsequently used to analyze the presence
of bomapin antigen in control and agonist-treated THP-1 cells.
Figure 4 shows a representative experiment in which 3-day control or treated THP-1 cytosol samples were
immunoprecipitated with either Sepharose-antibomapin or
Sepharose-normal rabbit IgG, and the immunoprecipitates analyzed
immunoblotting using biotin-labeled affinity purified antibomapin. A
major band at Mr 40 kD was detected in the control THP-1 samples
immunoprecipitated with the Sepharose-antibomapin (lane 1), which was
markedly decreased in intensity in cytosol prepared from cells
incubated for 3 days with PMA (lane 3).
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| Fig 4.
Detection of bomapin antigen in control and PMA- or
TNF--treated THP-1 cells. THP-1 cells (106 cells/mL, 25 mL/flask, 162 cm2/flask) were incubated in serum-containing
media in the absence (lanes 1 and 2) or presence of either PMA
(108 mol/L; lanes 3 and 4) or TNF- (30 U/mL; lanes 5 and 6) as described above. After 72 hours, the cells were washed twice,
homogenized, and centrifuged, and the cytosol preparations (1 mg) were
incubated with either Sepharose-antibomapin (lanes 1, 3, and 5) or
Sepharose-normal rabbit IgG (lanes 2, 4, and 6). The beads were washed
and the material eluting with SDS-sample buffer was analyzed by
immunoblotting using biotin-labeled affinity purified antibomapin,
streptavidin-peroxidase, and the enhanced
chemiluminescence detection system. Lane 7 contained 300 ng of GST-bomapin.
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DISCUSSION |
This report provides data indicating that the expression of bomapin
mRNA is significantly higher in the bone marrow than in peripheral
blood from normal individuals (Fig 1, Table 1). This observation is
consistent with the detection of bomapin transcript specifically in the
bone marrow and its absence in all other analyzed human tissues,
including samples from the peripheral blood cell-rich spleen or
placenta that were analyzed in our initial survey of human tissues by
Northern blotting.12 In addition, this observation supports
the concept that bomapin is predominantly expressed in cells that,
under normal conditions, are either not released from the bone marrow
(eg, stromal cells) or that are released into the blood only after
differentiation (eg, hematopoietic progenitor cells). Relatively high
levels of bomapin transcript in peripheral blood leukocytes from
patients with acute monoblastic leukemia (AML M5) and CMML (Fig 1,
Table 1) suggest that bomapin may be expressed preferentially in
hematopoietic progenitor cells of monocytic lineage. This hypothesis is
supported by the detection of bomapin mRNA in two tissue culture cell
lines of monocytic differentiation (ie, THP-1 and AML-193 cells).
Bomapin mRNA levels are downregulated on differentiation of THP-1 and
AML-193 cells by induction with PMA or TNF-. Preliminary experiments
suggest that HL-60 and U937 cells also express bomapin, which can be
downregulated by treatment with PMA (R.R. Schleef, personal
observations). Taken together, our data raise the possibility that
bomapin expression increases during certain early stages of monocytic
commitment, which is followed by a decrease of expression levels during
later stages of the differentiation to monocytes/macrophages.
High levels of two other ov-serpins have been shown in peripheral blood
monocytes and tissue culture cell lines of monocytic differentiation:
(1) the expression of PAI-2 in peripheral blood monocytes and
monocyte-like cell lines (eg, U937) is significantly elevated after the
induction of differentiation towards a macrophage phenotype with PMA or
TNF-23-25 and (2) leukocyte elastase inhibitor is
expressed constitutively in U937 cells and increasingly in peripheral
blood monocytes during in vitro culture, again suggesting that
expression of this inhibitor by monocytes increases during the
maturation process towards macrophages.26 The
downregulation of bomapin expression after induction of differentiation in THP-1 and AML-193 cells suggests that these different protease inhibitors may play specific roles during distinct stages of cellular development. Another widely-expressed intracellular inhibitor (ie, CAP)
was noted by Scott et al27 to be absent from THP-1 cells
using Northern blotting analysis. We have also observed that CAP mRNA
levels in THP-1 cells are below the sensitivity of Northern blotting
(Riewald et al, unpublished observations), and our present data suggest
that transcripts for CAP are present at low levels that require a
PCR-based assay. Our observation that bomapin is highly expressed in
the peripheral blood cells isolated from a patient with AML M5 coupled
with the low levels of CAP transcripts in this individual's blood (Fig
1, lane 14) would also be consistent with the production of specific
inhibitory molecules to protect or neutralize proteases that might be
produced during a particular stage of development. Although bomapin,
CAP, and PAI-2 are all serpins with arginine in the
specificity-determining P1 position of the reactive site loop that
plays a role in the formation of complexes with proteases of
trypsin-like specificity,4,7,8,12 alignment of their
primary structure indicates that bomapin contains a 22-amino acid loop
including a cluster of charged amino acids between helices C and D in
its tertiary structure12 that is absent in CAP. PAI-2 is
the only serpin that contains an even longer (ie, 37 amino acids)
interhelical loop,4 and recent data by Jensen et
al28 suggest that this region serves as a protein-binding
domain that may be critical for the function of PAI-2. It is possible
that the interhelical loop in bomapin may play a role in defining its
interaction with a specific cellular protein that is expressed during
differentiation of hematopoietic cells along the monocytic lineage.
Thus, second site interactions on these three protease inhibitors may
provide a rationale for their production in distinct cell types or at a
particular stage of differentiation.
| |
FOOTNOTES |
Submitted March 26, 1997;
accepted October 10, 1997.
Supported by a grant from the National Institutes of Health (HL49563).
Address reprint requests to Raymond R. Schleef, PhD, Department of
Vascular Biology (VB-1), The Scripps Research Institute, 10550 North
Torrey Pines Rd, La Jolla, CA 92037.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
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Abstract
Introduction
Abstract