Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Clara cell secretory protein (CCSP) is an
inhibitor of secretory phospholipase
A2. It is produced by airway
epithelial cells and is present in airway secretions. Because
interferon (IFN)- can induce gene expression in airway epithelial
cells and may modulate the inflammatory response in the airway, it was
of interest to study the effect of this cytokine on epithelial cell
CCSP mRNA expression and CCSP protein synthesis. A human bronchial
epithelial cell line (BEAS-2B) was used for this study. CCSP mRNA was
detected by ribonuclease protection assay. IFN-
was found to
increase CCSP mRNA expression in a time- and dose-dependent manner. The CCSP mRNA level increased after IFN-
(300 U/ml) treatment for 8-36 h, with the peak increase at 18 h. Immunobloting of CCSP protein also demonstrated that IFN-
induced the synthesis and secretion of CCSP protein in a time-dependent manner. Nuclear run-on,
CCSP reporter gene activity assay, and CCSP mRNA half-life assay
demonstrated that IFN-
-induced increases in CCSP gene expression were mediated, at least in part, at the posttranscriptional level. The
present study demonstrates that IFN-
can induce increases in
steady-state mRNA levels and protein synthesis of human CCSP protein in
airway epithelial cells and may modulate airway inflammatory responses
in this manner.
cytokines; airway inflammation; airway secretion
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INTRODUCTION |
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CLARA CELL SECRETORY PROTEIN (CCSP) (10), also designated Clara cell 10-kDa protein (CC10) (37), Clara cell 16-kDa protein (3), Clara cell 17-kDa protein (41), or polychlorinated biphenyl (PCB)-binding protein (20), was first named from the apparent molecular mass of this protein in nonreducing SDS-PAGE (37). Recent evidence (4) indicates it is also identical to human urinary protein-1. CCSP protein consists of two identical subunits of 70 amino acids joined by two disulfide bonds (2, 24, 41). The cDNAs and the derived amino acid sequences of human CCSP show striking homology with the rat and murine CC10 and rabbit uteroglobin (1, 11, 30, 34, 35, 39). The CCSP in rat and human lungs is the counterpart of rabbit uteroglobin in these species (22). CCSP is expressed in many nonrespiratory organs (27, 43) and also is highly expressed in airway epithelial cells (10, 12, 33, 36, 37). Although the CCSP gene (12, 43), amino acid sequence (14, 26, 35), and distribution (27, 37) of this protein are known, its physiological function is still unclear. Because a markedly similar protein, uteroglobin, is reported to have immunosuppressive, anti-inflammatory, antiproteinase, anti-phospholipase A2 (PLA2), and progesterone-binding activities (6, 22, 34), it is postulated that the CCSP gene product might also be an immunomodulatory or protective protein in the airway. Mice homozygous for deletion of the uteroglobin gene have increased serum PLA2 activity and develop glomerulonephritis (45). In addition, CCSP can bind methylsulfonyl PCBs (20, 26) to inhibit secretory PLA2 activity.
Epithelia provide a protective barrier for many organs exposed to the external environment. It is now recognized that the pulmonary epithelium actively participates in the inflammatory response and is capable of releasing various mediators, which have the potential to modulate local inflammatory reactions (40). The airway epithelium may play an active role in initiating and modulating airway inflammation (31). CCSP expression is not limited to Clara cells of the airway; instead, CCSP may be expressed by cells along the conducting airway. Therefore, we were interested in studying CCSP gene expression and translation in human bronchial epithelial cells.
Interferons (IFN-, IFN-
, and IFN-
) are a family of potent
multifunctional cytokines produced in the course of viral infections or
during the inflammatory response. In addition to their antiviral properties, IFNs are also known to induce a variety of other
physiological responses including antiprotozoal, antiproliferation, and
immunoregulatory activities (17, 28, 32). IFN-
exerts its biological
functions via a distinct cell-surface receptor (42). Human airway
epithelial cells express IFN-
receptors and respond to IFN
stimulation with increased expression of intercellular adhesion
molecule-1, and, similarly, BEAS-2B cells, an immortalized human airway
epithelial cell line, respond to IFN-
stimulation with increased
intercellular adhesion molecule-1 expression (19). IFN-
also induces
PLA2 activity,
PLA2 gene expression, and protein
synthesis in a variety of cell lines (29, 44). An IFN-
response
element has been identified in the 5' promoter region of the
murine CCSP gene (21). Because IFN-
can induce gene expression in
airway epithelial cells and may modulate the inflammatory response in
the airway, it was of interest to study the effect of this cytokine on
epithelial cell CCSP mRNA expression and CCSP protein synthesis.
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METHODS |
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Cell culture. BEAS-2B cells, a human bronchial epithelial cell line transformed by an adenovirus 12-SV40 virus hybrid, were a gift from Dr. J. E. Lecher and C. Harris (National Cancer Institute, Bethesda, MD). The cells were maintained in serum-free, hormonally defined culture medium (LHC-8, Biofluids, Rockville, MD) and grown in 175-cm2 tissue culture flasks (Nalge Nunc, Naperville, IL) that were precoated with a thin layer of rat tail type I collagen (Collaborative Research, Bedford, MA). Experiments were performed when the cells reached 80% confluence (~3 million cells/flask).
Ribonuclease protection assay. The
cells were treated with IFN- (10, 100, 300, and 1,000 U/ml;
Boehringer Mannheim, Indianapolis, IN) for 4-36 h. Total cellular
RNA was extracted from 175-cm2
culture flasks by the single-step guanidinium
thiocyanate-phenol-chloroform extraction method (Tri-Reagent, Molecular
Research, Cincinnati, OH). The RNA pellet was precipitated with
isopropanol and washed with 70% ethanol, then redissolved in diethyl
pyrocarbonate water. To construct the probe for CCSP mRNA,
a 367-bp product of CCSP cDNA was amplified by PCR with the following
sets of sense and antisense primers: 5' primer,
5'-CTCCACCATGAAACTCGCTG-3' (bases 1-20) and
3'primer, 5'-GAAGAGAGCAAGGCTGGTGG-3' (bases
367-348) (Bio-Synthesis, Lewisville, TX). The product of the CCSP
gene (bases 1-367) was cloned into the TA cloning vector
(Invitrogen, San Diego, CA). Orientation of the insert was determinated
by DNA sequencing with the dideoxynucleotide chain termination method. The CCSP and glyceradehyde-3-phosphate dehydrogenase (GAPDH; Ambion, Austin, TX) RNA probes were prepared by in vitro transcription using T7
polymerase with
[
-32P]CTP. A
ribonuclease protection assay (RPA) kit (RPAII, Ambion) was used for
the following procedures. Hybridization was performed at 45°C for
16 h and with 10 (for GAPDH) or 50 (for CCSP) µg of total RNA and
104 (for GAPDH) and 2 × 104 (for CCSP) counts/min of the
32P-labeled RNA probe. After
hybridization, the unhybridized RNA was digested by the addition of
diluted (1:150) RNase A-T1 mix at 37°C for 60 min. Digestion was
terminated by the addition of an RNase inactivation and precipitation
mixture. The protected RNA fragment was analyzed by autoradiography
after separation on 6% polyacrylamide-8 M urea gels.
Immunoblot of CCSP protein. The
BEAS-2B cells were grown in
175-cm2 flasks and treated with
IFN- (300 U/ml; Boehringer Mannheim); the cell culture media of
control and treated BEAS-2B cells were collected after IFN-
treatment for 8 and 18 h. After the culture media were dialyzed with a
3,500-molecular-weight membrane (Baxter, McGaw Park, IL) against
distilled water, 100 ml of the supernatant were concentrated to 0.5 ml
by lyophilization. Total protein was assayed with bicinchoninic acid
(BCA Reagent, Pierce, Rockford, IN). Samples containing 10 µg of
total protein were separated on 18% polyacrylamide gels (Novex, San
Diego, CA) under reducing conditions for 5 h. Another experiment was
done to verify the specific effect of IFN-
by treating the BEAS-2B
cells with IFN-
(300 U/ml; Boehringer Mannheim), interleukin (IL)-4
(40 ng/ml), and IL-13 (10 ng/ml; R&D Systems, Minneapolis, MN); the
cell culture media of control and treated BEAS-2B cells were collected
after treatment with the different cytokines for 18 h. After dialysis with a 3,500-molecular-weight membrane (Baxter) against distilled water, 25 ml of the culture media were concentrated to 1 ml by lyophilization. Total protein was assayed with the BCA Reagent (Pierce). Samples containing 25 µg of total protein were separated on
18% polyacrylamide gels (Novex) under reducing conditions for 5 h. The
separated proteins were electrophoretically transferred onto a
nitrocellulose membrane (Schleicher & Schuell, Keene, NH), then blocked
with 5% nonfat dry milk overnight. CCSP protein expression was
detected by using a 1:500 dilution of a rabbit anti-human CC10
polyclonal antibody (37) (a gift from Dr. Gurmukh Singh, Veterans
Affairs Medical Center, Pittsburgh, PA) followed by a 1:5,000 dilution
of horseradish peroxidase-conjugated goat anti-rabbit IgG as the second
antibody (Jackson ImmunoResearch Laboratories, West Grove, PA)
and developed with the enhanced chemiluminescence Western
blotting detection system (Amersham, Arlington Heights, IL).
Nuclear run-on assay. Nuclear run-on
assay was performed with a modification of previously described methods
(9, 15). The cells were stimulated with IFN- (300 U/ml) for 0, 0.5, 2, and 4 h. The cells were harvested after digestion with 0.1%
collagenase in Hanks' balanced salt solution without Ca2+
or Mg2+ [HBSS(
)] for 5-10 min.
After being washed with cold HBSS(
), the cell pellet was
resuspended with 4 ml of lysis buffer (10 mM Tris-buffered saline,
0.5% Nonidet P-40, 100 µg/ml of leupeptin, 50 µg/ml of aprotinin,
1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride),
incubated on ice for 5 min, and centrifuged at 500 g for 5 min. The nuclear pellet was
resuspended in 1 ml of lysis buffer and spun at 500 g for 5 min. The nuclei were
resuspended with 200 µl of reaction buffer [10 mM
Tris · HCl, pH 8.0; 5 mM MgCl2; 300 mM KCl; 1 mM
dithiothreitol; 0.5 mM each ATP, CTP, and GTP; and 200 µCi of
[
-32P]UTP (3,000 Ci/mmol; Amersham)] and then reacted at 30°C for 1 h. RNA was
extracted by the single-step guanidinium thiocyanate-phenol-chloroform extraction method with the addition of 100 µg of yeast transfer RNA.
The samples were resuspended to equal counts per minute per milliliter
(5-6 × 106 dpm/ml) in
hybridization buffer (50 mM PIPES, pH 6.8, 10 mM EDTA, 600 mM NaCl, and
0.2% SDS). The samples were allowed to hybridize to denatured DNA
targets (10 µg) that were slot blotted on nitrocellulose filters at
65°C for 40 h after prehybridization at 70°C for 2 h in
hybridization buffer containing 1% SDS. The DNA targets included the
linearized plasmid PCR II containing human cytosolic
PLA2 (cPLA2) cDNA as a positive
control, CCSP cDNA and GAPDH as an internal control, or the plasmid PCR
II as a negative control. At the end of the hybridization period, the
filters were washed in 2× saline-sodium citrate (SSC; 1×
SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.2% SDS,
1× SSC-0.2% SDS, and 0.5× SSC-0.2% SDS, respectively, at
65°C for 1 h and evaluated by autoradiography.
Plasmid construction for reporter
plasmids. Analysis of the nucleotide subsequences of
the 5' portion of the CCSP gene was performed in MacVector
Version 5.0.2. The 5'-flanking sequences of the CCSP gene were
generated by PCR from a human genomic DNA library with primers derived
from a published sequence (EMBL accession no. X59875). A common 24-mer
3' primer was synthesized for all the constructs, which
corresponds from bases +31 to +13 of the sequence of human CCSP gene
and contains a Bgl II restriction site
at its 5'-end (5'-GAAGATCTTCTCTGGTTCCGTTCTCTG-3').
Individual 5' primers were constructed to generate various
deletion constructs. Different 24-mer 5' primers were
synthesized, which contain a 5'
Kpn I site and started at base
801 (5'-GGGGTACCAGAATAAACATCTAAAGA-3') and base
168 (5'-GGGGTACCTGGGGACAGAAACTGGGT-3'),
respectively. The different truncated promoters (bases
801 to
+31 and
168 to +31) were cloned upstream from the firefly
luciferase coding region in the pGL3-basic vector (Promega, Madison,
WI).
Transient transfection of reporter
plasmids. BEAS-2B cells were maintained at 37°C
under 5% CO2 in LHC-8 medium
(Biofluids). For each transfection experiment, the cells were seeded in
six-well plates and transfected with 2 µg of reporter plasmid DNA
with Lipofectamine Reagent (Life Technologies, Gaithersburg, MD) when the cells reached 80% confluence. As a control for transfection efficiency, 0.2 µg of a secreted placental alkaline phosphatase (SEAP) plasmid (pCMV/SEAP Vector; Tropix, Bedford, MA) was added. After
a 2-h transfection period, the cells were placed in culture medium and
incubated for 16 h. The cells were then treated with IFN- (300 U/ml)
for 8-18 h, the transfected cells were harvested and lysed, and
the extracts were evaluated with luciferase assay reagent (Promega) in
a luminometer (model 2010, Analytical Luminescence Laboratories, Ann
Arbor, MI). SEAP activity was assayed in culture media with a SEAP
assay kit (Tropix).
Measurement of CCSP mRNA half-life in BEAS-2B
cells. BEAS-2B cells were grown in
175-cm2 flasks for the
determination of CCSP mRNA half-life in control cells or in cells after
IFN- stimulation. Control cells or cells stimulated with IFN-
(300 U/ml) for 18 h before the addition of actinomycin D (50 µg/ml;
Calbiochem, San Diego, CA) were harvested for RNA at 0, 8, 18, and 24 h
after the addition of actinomycin D. Total RNA was prepared, and RPAs
were performed as described in Ribonuclease protection
assay. The protected fragments were quantitated with a densitometer (Molecular Dynamics, Sunnyvale, CA).
The quantity of CCSP mRNA was normalized to the amount of GAPDH by
calculating a CCSP-to-GAPDH ratio for each sample. All time points were
performed in triplicate.
Statistical analysis of the mRNA half-life was performed by ANOVA in Microsoft Excel, Version 5.0.
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RESULTS |
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BEAS-2B cells produce CCSP mRNA and IFN- increases
CCSP steady-state mRNA levels. To determine whether
BEAS-2B cells produce CCSP mRNA, steady-state levels of CCSP mRNA were
measured by RPA of total cellular RNA extracted from cells that were
incubated without or with IFN-
(300 U/ml) for 4-36 h. As shown
in Fig. 1, these cells produce CCSP mRNA,
and the steady-state level of CCSP mRNA is significantly induced by
IFN-
treatment over 8-36 h.
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To determine a dose-related effect of IFN- on the changes in
steady-state CCSP mRNA, cells were incubated with 10-1,000 U/ml of
IFN-
for 24 h. Total cellular RNA was extracted, and an RPA for CCSP
mRNA was performed. IFN-
in concentrations of 100-1,000 U/ml
induced a dose-related change in CCSP mRNA (Fig.
2).
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IFN- induces CCSP protein production and release in
BEAS-2B cells. To determine the effect of IFN-
treatment on the level of CCSP synthesized and released into the cell
culture medium, BEAS-2B cells were treated with IFN-
(300 U/ml) for
8 and 18 h, the time points at which CCSP mRNA levels significantly
increased after IFN-
stimulation. The culture media were
concentrated and subjected to polyacrylamide gel electrophoresis
followed by Western blotting. IFN-
treatment increased the release
of CCSP immunoreactive material into the supernatant at both 8 and 18 h
(Fig. 3). This material had an apparent
molecular mass of ~7 kDa on polyacrylamide gel electrophoresis.
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Additional experiments were performed to determine whether IFN- has
a specific effect on CCSP synthesis and release. BEAS-2B cells were
treated with IFN-
(300 U/ml) or with the TH2 cytokine IL-4 (40 ng/ml) or IL-13 (10 ng/ml) for 18 h, the time point at which CCSP mRNA
levels significantly increased after IFN-
stimulation. The culture
media were concentrated and subjected to polyacrylamide gel
electrophoresis followed by Western blotting. IFN-
treatment increased the release of CCSP immunoreactive material into the cell
culture medium at 18 h; neither IL-4 nor IL-13 treatment had an effect
(Fig. 4).
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IFN- regulates CCSP gene expression at
posttranscriptional level. To further understand the
mechanism involved in CCSP gene regulation by IFN-
, three studies
were performed. The investigation of CCSP promoter activation by
IFN-
was done by transfecting two different constructs that
contained the upstream sequence of the CCSP promoter into BEAS-2B
cells, followed by stimulation with IFN-
. Although both reporter
genes exhibited promoter activity, IFN-
treatment of cells for 8 or
18 h resulted in no increase in luciferase activity above baseline
(Fig. 5). Second, control of CCSP
steady-state mRNA levels was studied with a transcriptional assay. An
in vitro transcriptional assay was performed after IFN-
treatment
for 0.5-4 h. Although the
cPLA2 gene transcriptional rate
was increased after treatment for 0.5-4 h, no increase was observed in the nuclear transcription rate of the CCSP gene over the
period studied (Fig. 6). Therefore, both
assays did not demonstrate changes in CCSP mRNA transcription. Because
these two assays for transcription did not suggest transcriptional
control of the observed changes in CCSP mRNA, the half-life of CCSP
mRNA was studied. Total RNA was extracted from control and
IFN-
-stimulated cells treated with the inhibitor of RNA synthesis,
actinomycin D, as described in
METHODS. CCSP mRNA levels were
analyzed by RPA. As shown in Fig. 7,
IFN-
-stimulated cells displayed a prolonged stability of CCSP mRNA
compared with the unstimulated control cells. While the calculated
half-life of CCSP mRNA in control cells was ~15 h, the calculated
half-life of CCSP mRNA in the IFN-treated cells was prolonged to ~40
h (P < 0.01). Therefore, in airway
epithelial cells, IFN-
increased the steady-state level of CCSP
mRNA, at least in part, at a posttranscriptional level.
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DISCUSSION |
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The expression of CCSP protein in the airway epithelium has been previously reported (10, 11, 27, 34, 37). Human CCSP protein has striking structural and sequence similarities with the rat and murine CCSPs and the uteroglobin gene product in the rabbit. The physiological role(s) of CCSP is still unclear. The human CCSP protein has been found to have several biological properties including inhibition of PLA2 activity (22, 23, 34), binding of PCBs, and reduction of foreign protein antigenicity by forming a complex with transglutaminase (22). It has been reported that CCSP has a clear inhibitory effect on PLA2 activity in lung fibroblasts in vitro (16). CCSP was able to inhibit fibroblast chemotaxis in vitro by mechanisms that may be related to the inhibition of PLA2 activity. Significantly higher levels of CCSP have been observed in bronchoalveolar lavage fluid from patients with acute lung injury (13). The inhibition of PLA2 by CCSP may be important in controlling extracellular inflammatory activity in the lung. Inhibition of PLA2 activity might result in a reduction in the metabolism of arachidonic acid and decrease the production of lipid mediators of inflammation.
The results of our experiments indicate that human bronchial epithelial
cells in culture contain detectable levels of CCSP mRNA and can
synthesize and secrete CCSP. The secretion of CCSP protein is induced
by IFN- but not by the TH2 cytokines IL-4 and IL-13. For Western
blot studies, we have used an antibody utilized in a previous study of
human CCSP (37). In our studies, the secreted CCSP migrated on SDS-PAGE
under reducing conditions with an apparent molecular mass of ~7 kDa.
This is in agreement with the Clara cell protein size reported by
Bernard and colleagues (3, 4). They analyzed CCSP by electron
spray-mass spectrometry to determine the extact size of this protein.
Their results suggested that the CCSP has a 15.8-kDa molecular mass and
is 7.8 kDa after reduction of the protein.
IFN treatment may alter the rates of synthesis and the steady-state
level of a variety of cellular mRNAs and the corresponding proteins
(32). Although IFNs can increase transcription rates within minutes
after exposure of the cells to IFN and the steady-state level of
IFN-inducible mRNAs may be primarily regulated at the transcriptional
level, posttranscriptional regulation may also be involved (8, 38).
Examples of posttranscriptional regulation of mRNA have been well
documented, particularly at the level of mRNA stability (18, 25, 28).
Friedman et al. (8) found that the mRNA of one of the genes they cloned
from IFN--induced T98G neuroblastoma cells (designated mRNA
1-8) continued to accumulate after 8-12 h of IFN-
stimulation, whereas the transcription rate began to fall after
4-6 h. Our study showed that IFN-
may alter the rate of
synthesis of CCSP protein at a posttranscriptional level. We observed
that an increase in steady-state CCSP mRNA paralleled the increased
protein level in human bronchial epithelial cells after IFN-
stimulation. Although there are two areas in the 5' promoter
region of the CCSP gene that might be putative [IFN-
response
element consensus sequence sites (5) at base
758
(10 of 14 nucleotides) and at base
148 (10 of 14 nucleotides)], the reporter gene constructs (bases
801 to
+31 and
168 to +31) contained both of these sites, and the
luciferase activity measured in the different reporter gene constructs
did not increase after IFN-
treatment. Our data also showed an
increase in CCSP mRNA after 8-36 h of treatment with IFN-
despite no change in the transcription rate after IFN-
stimulation.
This suggests that the stimulatory effects of IFN-
on human CCSP
gene expression in BEAS-2B cells is controlled, at least in part, at
the posttranscriptional level.
Dierynck et al. (7) found that CCSP potentially inhibits IFN-
production and biological activity in peripheral blood mononuclear cells. We observed that recombinant human IFN-
can stimulate CCSP
gene expression and protein synthesis in cultured human bronchial epithelial cells. Therefore, IFN-
may modulate CCSP production, and
CCSP may feed back to modulate IFN-
production by peripheral blood
lymphocytes. This loop may represent another example of cytokine
modulation of epithelial cell function and epithelial cell modulation
of the inflammatory response in the lung.
Airway epithelial cells are increasingly recognized for their ability
to participate in the regulation of local inflammatory and immune
responses. Our present study demonstrates that IFN- can induce
increases in steady-state mRNA levels and protein synthesis of human
CCSP protein in airway epithelial cells and may modulate airway
inflammatory responses in this manner.
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FOOTNOTES |
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Address for reprint requests: J. H. Shelhamer, Bldg. 10, Rm. 7-D-43, NIH, Bethesda, MD 20892.
Received 30 June 1997; accepted in final form 1 February 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Andersson, O.,
L. Nordlund-Moller,
M. Bronnegard,
F. Sirzea,
E. Ripe,
and
J. Lund.
Purification and level of expression in bronchoalveolar lavage of a human polychlorinated biphenyl (PCB)-binding protein: evidence for a structural and functional kinship to the multihormonally regulated protein uteroglobin.
Am. J. Respir. Cell Mol. Biol.
5:
6-21,
1991[Medline].
2.
Bally, R.,
and
J. Delletre.
Structure and refinement of the oxidized P21 form of uteroglobin at 1.64 Å resolution.
J. Mol. Biol.
206:
153-170,
1989[Medline].
3.
Bernard, A.,
X. Dumont,
H. Roels,
R. Lauwerys,
I. Dierynck,
M. De Ley,
V. Stroobant,
and
E. de Hoffmann.
The molecular mass and concentrations of protein 1 or Clara cell protein in biological fluids: a reappraisal.
Clin. Chim. Acta
223:
189-191,
1993[Medline].
4.
Bernard, A.,
H. Roels,
R. Lauwerys,
R. Witters,
C. Gielens,
A. Soumillion,
J. Van Damme,
and
M. De Ley.
Human urinary protein 1: evidence for identity with the Clara cell protein and occurrence in respiratory tract and urogenital secretions.
Clin. Chim. Acta
207:
239-249,
1992[Medline].
5.
Dale, T.,
J. Rosen,
M. Guille,
A. Lewin,
A. Porter,
I. Kerr,
and
G. Stark.
Overlapping sites for constitutive and induced DNA binding factors involved in interferon-stimulated transcription.
EMBO J.
8:
831-839,
1989[Abstract].
6.
Davidson, F. F.,
and
E. A. Dennis.
Biological relevance of lipocortins and related proteins as inhibitors of phospholipase A2.
Biochem. Pharmacol.
38:
3645-3651,
1989[Medline].
7.
Dierynck, I.,
A. Bernard,
H. Roels,
and
M. De Ley.
Potent inhibition of both human interferon- production and biologic activity by the Clara cell protein CC16.
Am. J. Respir. Cell Mol. Biol.
12:
205-210,
1995[Abstract].
8.
Friedman, R. L.,
S. P. Manly,
M. McMahon,
I. M. Kerr,
and
G. R. Stark.
Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells.
Cell
38:
745-755,
1984[Medline].
9.
Greenberg, M. E.,
and
E. B. Ziff.
Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene.
Nature
311:
433-438,
1984[Medline].
10.
Hackett, B. P.,
N. Schimizu,
and
J. D. Gitlin.
Clara cell secretory protein gene expression in bronchiolar epithelium.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L399-L404,
1992
11.
Hagen, G.,
M. Wolf,
S. L. Katyal,
G. Singh,
M. Beato,
and
G. Suske.
Tissue-specific expression, hormonal regulation and 5'-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobin.
Nucleic Acids Res.
18:
2939-2946,
1990[Abstract].
12.
Hay, J. G.,
C. Danel,
C. S. Chu,
and
R. G. Crystal.
Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L565-L575,
1995
13.
Jorens, P. G.,
Y. Sibille,
N. J. Goulding,
F. J. Van Overveld,
A. G. Herman,
L. Bossaert,
W. A. De Backer,
R. Lauwerys,
R. J. Flower,
and
A. Bernard.
Potential role of Clara cell protein, an endogenous phospholipase A2 inhibitor, in acute lung injury.
Eur. Respir. J.
8:
1647-1653,
1995
14.
Katyal, S. L.,
G. Singh,
W. E. Brown,
A. L. Kennedy,
N. Squeglia,
and
M. L. Wong Chong.
Clara cell secretory (10 kDa) protein: amino acid and cDNA nucleotide sequences and developmental expression.
Prog. Respir. Res.
25:
29-35,
1990.
15.
Kavanaugh, W. M.,
G. R. Harsh IV,
N. F. Starksen,
C. M. Rocco,
and
L. T. Williams.
Transcriptional regulation of the A and B chain genes of platelet-derived growth factor in microvascular endothelial cells.
J. Biol. Chem.
263:
8470-8472,
1988
16.
Lesur, O.,
A. Bernard,
K. Arsalane,
R. Lauwerys,
R. Begin,
A. Cantin,
and
D. Lane.
Clara cell protein (CC16) induces a phospholipase A2-mediated inhibition of fibroblast migration in vitro.
Am. J. Respir. Crit. Care Med.
152:
290-297,
1995[Abstract].
17.
Lewis, D. B.,
and
C. B. Wilson.
Gamma-interferon: an immunoregulatory lymphokine with immunotherapeutic potential.
Pediatr. Infect. Dis. J.
9:
642-651,
1990[Medline].
18.
Levy, A. P.,
N. S. Levy,
and
M. A. Goldberg.
Post-transcription regulation of vascular endothelial growth factor by hypoxia.
J. Biol. Chem.
271:
2746-2753,
1996
19.
Look, D. C.,
S. Rapp,
B. Keller,
and
M. J. Holtzman.
Selective induction of intercellular adhesion molecule-1 by interferon- in human airway epithelial cells.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L79-L87,
1992
20.
Lund, J.,
L. Nordlund,
and
J. Gustafsson.
Partial purification of a binding protein for polychlorinated biphenyls from rat lung cytosol: physiochemical and immunochemical characterization.
Biochemistry
27:
7895-7901,
1988[Medline].
21.
Magdaleno, S. M.,
G. Wang,
K. J. Jackson,
M. K. Ray,
S. Welty,
R. H. Costa,
and
F. J. DeMayo.
Interferon- regulation of Clara cell gene expression: in vivo and in vitro.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L1142-L1151,
1997
22.
Mantile, G.,
L. Miele,
E. Cordella-Miele,
G. Singh,
S. L. Katyal,
and
A. B. Mukherjee.
Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin.
J. Biol. Chem.
268:
20343-20351,
1993
23.
Miele, L.,
E. Cordella-Miele,
A. Facchiano,
and
A. B. Mukherjee.
Novel anti-inflammatory peptides from the region of highest similarity between uteroglobin and lipocortin I.
Nature
335:
726-729,
1988[Medline].
24.
Morize, I.,
E. Surcouf,
M. C. Vaney,
Y. Epelboin,
M. Buehner,
F. Fridlansky,
E. Milgrom,
and
J. P. Mornon.
Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 Å resolution.
J. Mol. Biol.
194:
725-739,
1987[Medline].
25.
Nicolini, G.,
M. Miloso,
M. C. Moroni,
L. Beguinot,
and
L. Scotto.
Post-transcriptional control regulates transforming growth factor in the human carcinoma KB cell line.
J. Biol. Chem.
271:
30290-30296,
1996
26.
Nordlund-Moller, L.,
O. Andersson,
R. Ahlgren,
J. Schilling,
M. Gillner,
J. A. Gustafsson,
and
J. Lund.
Cloning, structure, and expression of a rat binding protein for polychlorinated biphenyls. Homology to the hormonally regulated progesterone binding protein, uteroglobin.
J. Biol. Chem.
265:
12690-12693,
1990
27.
Peri, A.,
E. Cordella-Miele,
L. Miele,
and
A. B. Mukherjee.
Tissue-specific expression of the gene coding for human Clara cell 10-kD protein, a phospholipase A2-inhibitory protien.
J. Clin. Invest.
92:
2099-2109,
1993[Medline].
28.
Pestka, S.,
L. A. Langer,
K. C. Zoon,
and
C. E. Samuel.
Interferons and their actions.
Annu. Rev. Biochem.
56:
727-777,
1987[Medline].
29.
Ponzoni, M.,
P. G. Montaldo,
and
P. Cornaglia-Ferraris.
Stimulation of receptor-coupled phospholipase A2 by interferon-.
FEBS Lett.
310:
17-21,
1992[Medline].
30.
Ray, M. K.,
S. Magdaleno,
B. M. O'Malley,
and
F. J. DeMayo.
Cloning and characterization of the mouse Clara cell specific 10 kDa protein gene: comparison of the 5'-flanking region with human rat and rabbit gene.
Biochem. Biophys. Res. Commun.
197:
163-171,
1993[Medline].
31.
Rennard, S. I.,
J. D. Beckmann,
and
R. A. Robbins.
Biology of airway epitheilial cells.
In: The Lung: Scientific Foundations, edited by R. G. Crystal,
and J. B. West. New York: Raven, 1991, p. 157-167.
32.
Sen, G. C.,
and
P. Lengyel.
The interferon system: a bird's eye view of its biochemistry.
J. Biol. Chem.
267:
5017-5020,
1992
33.
Singh, G.,
and
S. L. Katyal.
An immunological study of the secretory products of rat Clara cells.
J. Histochem. Cytochem.
32:
49-54,
1984[Abstract].
34.
Singh, G.,
S. L. Katyal,
W. E. Brown,
A. L. Kennedy,
U. Singh,
and
M. L. Wong-Chong.
Clara cell 10 kDa protein (CC10): comparison of structure and function to uteroglobin.
Biochim. Biophys. Acta
1039:
348-355,
1990[Medline].
35.
Singh, G.,
S. L. Katyal,
W. E. Brown,
S. Phillips,
A. L. Kennedy,
J. Anthony,
and
N. Squeglia.
Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein.
Biochim. Biophys. Acta
950:
329-337,
1988[Medline].
36.
Singh, G.,
S. Singal,
S. L. Katyal,
W. E. Brown,
and
S. A. Gottron.
Isolation and amino acid composition of the isotope of a rat Clara cell specific protein.
Exp. Lung Res.
13:
299-309,
1987[Medline].
37.
Singh, G.,
J. Singh,
S. L. Katyal,
W. E. Brown,
J. A. Kramps,
I. L. Paradis,
J. H. Dauber,
T. A. MacPherson,
and
N. Squeglia.
Identification, cellular localization, isolation and characterization of human Clara cell-specific 10 kD protein.
J. Histochem. Cytochem.
36:
73-78,
1988[Abstract].
38.
Sivo, J.,
A. D. Politis,
and
S. N. Vogel.
Differential effects of interferon- and glucocorticoids on Fc
R gene expression in murine macrophages.
J. Leukoc. Biol.
54:
451-457,
1993[Abstract].
39.
Suske, G.,
M. Wenz,
A. C. Cato,
and
M. Beato.
The uteroglobin gene region: hormonal regulation, repetitive elements and complete nucleotide sequence of the gene.
Nucleic Acids Res.
11:
2257-2271,
1983[Abstract].
40.
Thompson, A. B.,
R. A. Robbins,
D. J. Romberger,
J. H. Sisson,
J. R. Spurzen,
H. Teschler,
and
S. I. Rennard.
Immunological functions of the pulmonary epithelium.
Eur. Respir. J.
8:
127-149,
1995
41.
Umland, T. C.,
S. Swaminathan,
W. Furey,
G. Singh,
J. Pletcher,
and
M. Sax.
Refined structure of rat Clara cell 17 kD protein at 3.0 Å resolution.
J. Mol. Biol.
224:
441-448,
1992[Medline].
42.
Van Loon, A. P.,
L. Ozmen,
M. Fountonlakis,
M. Kania,
M. Haiker,
and
G. Garotta.
High-affinity receptor for interferon-gamma (IFN-), a ubiquitous protein occurring in different molecular forms on human cells: blood monocytes and eleven different cell lines have the same IFN-
receptor protein.
J. Leukoc. Biol.
49:
462-473,
1991[Abstract].
43.
Wolf, M.,
J. Klug,
R. Hackenberg,
M. Gessler,
K. H. Grzeschik,
M. Beato,
and
G. Suske.
Human CC10, the homologue of rabbit uteroglobin: genomic cloning, chromosomal localization and expression in endometrial cell lines.
Hum. Mol. Genet.
1:
371-378,
1992[Abstract].
44.
Wu, T.,
S. J. Levine,
M. G. Lawrence,
C. Logun,
W. Angus,
and
J. H. Shelhamer.
Interferon- induces the synthesis and activation of cytosolic phospholipase A2.
J. Clin. Invest.
93:
571-577,
1994[Medline].
45.
Zhang, Z.,
G. Kundu,
C. Yuan,
J. Ward,
E. Lee,
F. DeMayo,
H. Westphal,
and
A. Mukherjee.
Severe fibronectin-deposit renal glomerular disease in mice lacking uteroglobin.
Science
276:
1408-1412,
1997