(Received for publication, December 14, 1994; and in revised form, April 28, 1995)
From the
Pulmonary surfactant protein C (SP-C) is a 3.7-kDa, hydrophobic
peptide secreted by alveolar type II epithelial cells. SP-C enhances
surface tension lowering activity of surfactant phospholipids that is
critical to the maintenance of alveolar volume at end expiration. The
proinflammatory cytokine, tumor necrosis factor (TNF-
),
decreased SP-C mRNA within 24 h of intratracheal administration to
mice. In vitro, TNF-
decreased SP-C mRNA in a time- and
dose-dependent manner, reducing the steady state levels of SP-C mRNA by
3-5-fold. In contrast, TNF-
induced intercellular adhesion
molecule-1 expression in both mouse lung and murine lung epithelial
cell lines. Nuclear run-on analysis demonstrated that transcription of
both the endogenous SP-C gene and a human SP-C promoter-driven
transgene was inhibited by TNF-
. TNF-
decreased mouse
SP-C-chloramphenicol acetyltransferase mRNA in stably transfected
murine lung epithelial cells. Deletion analysis of the SP-C promoter
region demonstrated that TNF-
inhibited gene expression in
constructs containing 320 base pairs 5` from the start of transcription
of the mouse SP-C gene. Inhibition of surfactant protein C gene
transcription by TNF-
may contribute to the abnormalities of
surfactant homeostasis associated with pulmonary injury and infection.
Acute respiratory distress syndrome (ARDS) ()is a
common, often fatal complication of infection, shock, and trauma and
has been associated with increased levels of the proinflammatory
cytokine, TNF-
(1) . TNF-
and other cytokines increase
the expression of adhesion molecules including intercellular adhesion
molecule 1 (ICAM-1), vascular cell adhesion molecule 1, and endothelial
leukocyte adhesion molecule 1 and alter vascular permeability following
injury and infection (reviewed in (2) ). The resulting
pulmonary inflammation and increased alveolar permeability cause
leakage of serum proteins into the alveolar space leading to surfactant
dysfunction and loss of lung compliance. The hydrophobic proteins, SP-B
and SP-C, enhance the rate of spreading and stability of surfactant
phospholipids and protect against surfactant inactivation by serum
proteins (reviewed in (3) ). Surfactant proteins are decreased
in bronchoalveolar lavage of ARDS patients(4) , indicating that
the protective effects of surfactant proteins on respiratory function
may be compromised. In laboratory animals, intratracheal administration
of TNF-
causes increased pulmonary ICAM-1 expression, acute lung
inflammation, and pulmonary edema(5, 6, 7) . In vitro, TNF-
decreased SP-A and SP-B synthesis, the
latter mediated by destabilization of SP-B mRNA through element(s) in
the 3`-untranslated region of the human SP-B mRNA(8) . Effects
of TNF-
on SP-C gene expression have not been previously reported.
In the lung, TNF- is produced primarily by activated pulmonary
macrophages and accumulates in the bronchoalveolar lavage fluid of
patients with acute lung injury. Most cells express two distinct high
affinity receptors for TNF-
, TNF-R1 (type 1 or 55-kDa receptor)
and TNF-R2 (type 2 or 75-kDa receptor)(9, 10) .
Induction of ICAM-1 expression by TNF-
is mediated by TNF-R1
signaling as an early event after binding to endothelial
cells(11) . The pattern of ICAM-1 induction by cytokines
(TNF-
and interferon-
) in primary tracheal epithelial cells
is distinct from that in vascular endothelial cells (12) ,
supporting the concept that different intracellular pathways are
involved in TNF-
signaling in various lung cell types.
In the
present study, TNF- decreased SP-C mRNA in vivo and
inhibited SP-C gene transcription in vitro. Cis-active signals
located within 320 base pairs of the start of transcription of the
mouse SP-C gene were sufficient to mediate the inhibitory effects of
TNF-
on SP-C gene expression in respiratory epithelial cells.
MLE-15 cells
were co-transfected with CAT reporter constructs (described below) and
pRSVneo using the CaPO co-precipitation
method(16) . After 3 days of growth, transfectants were
selected by treatment with 400 µg/ml G418 (Life Technologies, Inc.)
in HITES medium with 2% fetal bovine serum. Pools of clones containing
several hundred colonies were trypsinized and treated as mixed
populations. At least two independently generated mixed populations
were tested for each construct.
The S1 probe specific for
murine SP-C mRNA (20) contained the 3`-half of the SP-C gene
and was linearized at the BamHI site 135 nucleotides 3` of the
intron-exon junction of exon 6. A portion of the murine cytoplasmic
-actin cDNA(20) , ScaI to the 3` end, was
subcloned in pGEM7z (f+), and a 1.1-kb Alw44I fragment of
this subclone, containing 100 bp of
-actin cDNA, was used as an
internal control. In experiments with mouse lung RNA, a probe for mouse
ribosomal protein L32 (L32) cDNA was used as an internal control since
TNF-
treatment caused a slight induction of
-actin mRNA in vivo. A 0.6-kb HindIII to DraI fragment
encompassing most of the L32 cDNA was subcloned from pL32/33 (22) into pGEM7z(f+). A 730-bp PvuII fragment of
the subclone containing 400 bp of L32 cDNA was used as a probe for S1
nuclease analysis, and the whole insert (EcoRI to BamHI) was used for Northern blots.
For Northern blot
analysis, 15 µg of total RNA/lane was resolved by electrophoresis
through 1% agarose, 2.2 M formaldehyde gels. RNAs were blotted
onto Magna charge nylon membranes (MSI, Westborom, MA), UV
cross-linked, and sequentially hybridized to
-
P-labeled probes, as indicated, in hybridization
buffer (0.5 M phosphate buffer at pH 7.2, 3.5% SDS, 33%
formamide, 1 mg/ml bovine serum albumin, 1 mM EDTA, 20
µg/ml yeast tRNA) at 55 °C overnight. Blots were washed to 0.2
SET (1
SET is 150 mM NaCl, 30 mM Tris-HCl, pH 7.8, 2 mM EDTA), 0.1% SDS at 55 °C,
autoradiographed, and quantitated by a PhosphorImager.
To label nascent transcripts, nuclei
were thawed on ice and incubated in 25 mM HEPES (pH 7.4), 2.5
mM MgCl, 2.5 mM dithiothreitol, 75
mM KCl, and 5% glycerol in the presence of 0.35 mM
each ATP, GTP, CTP, and 0.4 µM UTP plus 200 µCi of
[
-
P]UTP (3000 Ci/mM, DuPont NEN)
for 30 min at 30 °C. The reaction was stopped by digestion with
RNase-free DNase I for 15 min at 37 °C followed by digestion with
proteinase K in the presence of 0.1% SDS and extraction with
phenol/chloroform. Nucleic acids were precipitated with cold 10%
trichloroacetic acid, sequentially washed with 5% trichloroacetic acid
and ethanol, and resuspended in 10 mM Tris-HCl (pH 7.4), 1
mM EDTA, and 0.5% SDS. As a control for specificity of
transcription, one reaction was performed in the presence of 2
µg/ml
-amanitin to inhibit RNA polymerase II. Hybridization of
run-on transcription products to single-stranded M13-based probes bound
to nylon filters was performed at 55 °C for 48-56 h in the
Northern hybridization buffer described above using at least 10
cpm/ml.
Figure 1:
SP-C and
ICAM-1 mRNA after intratracheal administration of murine or human
TNF-. Adult FVB/N mice were treated with a single dose of 50
µg of human or mouse TNF-
or normal saline. Total lung RNA was
harvested 24 h later. PanelA represents S1 nuclease
analysis of 2 µg of RNA per sample probed for both SP-C and L32
mRNAs, quantitated by a PhosphorImager, and plotted relative to L32
(see ``Materials and Methods''). The mean of the
saline-treated animals was set to 1.0 and is indicated by a dashedline. Each point represents the relative mRNA
level from an individual animal (n = 1-4
measurements/animal). PanelB represents the relative
level of ICAM-1 mRNA expression in each animal as determined by
Northern blot analysis of 15 µg of lung RNA per lane hybridized sequentially with cDNA probes for ICAM-1 and L32. Blots
were quantitated by a PhosphorImager, and the ICAM-1 signal was
corrected by normalizing to L32 as in A. Statistical
differences between saline-treated and TNF-
-treated groups are
indicated:**, p < 0.0001; *, p = 0.01; #, p = 0.05 (Fisher's protected least significant
difference statistic).
Figure 2:
Murine and human TNF- inhibit SP-C
mRNA and induce ICAM-1 mRNA in vitro. MLE-12 cells were
treated with human or mouse TNF-
for 24 h, and total RNA was
isolated. PanelA represents an S1 nuclease assay for
SP-C and L32 mRNAs using 10 µg of RNA per lane. Lanes1 and 2 contain RNA from untreated cells, lane3 from cells treated with 30 ng/ml rmTNF-
, lane4 with 60 ng/ml rmTNF-
, lane5 with 30 ng/ml rhTNF-
, and lane6 with 60
ng/ml rhTNF-
. PanelB represents a Northern blot
of the same samples as in A, sequentially hybridized with
probes for TAg and ICAM-1 mRNA. Ribosomal protein L32 mRNA (L32) was used as a control for equal loading of lanes. Results are representative of two independent
experiments, each performed in duplicate.
Inhibitory effects of TNF- on murine SP-C
and SV40 TAg mRNAs were time- and dose-dependent. Maximal inhibition of
SP-C and SV40 TAg mRNAs was observed with 20-40 ng/ml rmTNF (Fig. 3). TNF-
significantly decreased SV40 TAg mRNA in
MLE-12 cells by 12 h of treatment and decreased endogenous SP-C mRNA at
all time points after 12 h (Fig. 4). In contrast, ICAM-1 mRNA
was maximally induced by 8 h and remained elevated 24 h after treatment
with TNF-
. TNF-
did not alter cell viability as assessed by
trypan blue exclusion and a non-radioactive cell proliferation assay
(Promega, Madison, WI). As observed in vivo, both rh- and
rmTNF-
significantly inhibited SP-C mRNA in vitro.
Figure 3:
TNF- inhibits endogenous SP-C and
transgenic human 3.7SP-C SV40 TAg mRNAs in vitro. MLE12 cells
were treated with increasing concentrations of rmTNF-
for 24 h,
and total RNA was isolated. Upperpanels represent S1
nuclease protection assays for SP-C and
-actin mRNAs loading 10
µg of RNA per lane. Lowerpanels represent Northern blots of 15 µg of the same RNAs as above,
sequentially hybridized with probes for SV40 TAg and
-actin mRNA
as an internal control. The identity of each band is indicated at the right. Lanes1-5 and 6-9 represent two independent experiments.
Figure 4:
Time course of the inhibitory effect of
TNF- on SP-C mRNA. MLE-12 cells were treated with 25 ng/ml
rmTNF-
, and total RNA was harvested at the indicated time points. Lane1, untreated; lane2, 8 h; lane3, 12 h; lane4, 15 h; lane5, 18 h; lane6, 21 h; lane7, 24 h. PanelA represents an
S1 nuclease analysis of 10 µg/lane for SP-C mRNA using
-actin
as an internal control. PanelB represents Northern
blot analysis of the same RNA samples (15 µg/lane) as in A, sequentially hybridized with probes for SV40 TAg,
-actin, and ICAM-1 mRNAs. Data representative of three experiments
are shown.
Figure 5:
TNF- inhibits transcription from the
mouse and human SP-C promoters in vitro. A, MLE-12
cells were treated for 8, 16, or 24 h with 25 ng/ml rmTNF-
. Nuclei
were harvested, and nascent transcripts were elongated in the presence
of [
-
P]UTP. 2
10
dpm/ml
were hybridized to slot blots of single-stranded probes for
-actin, SV40 TAg (TAg), murine SP-C, and M13 mp19 vector
DNA.
-Amanatin (
AM, 2 µg/ml) was used to inhibit
RNA polymerase II transcription. B, relative transcription
rates of mouse SP-C and human 3.7 SP-C-driven TAg. Three independent
nuclear run-on experiments were quantitated by using a PhosphorImager.
The relative transcription rates of mouse SP-C (
) and human 3.7
SP-C TAg (
) were determined by normalizing the signals to actin
and setting T
=
1.0.
Figure 6:
TNF- inhibits activity of the mouse
SP-C proximal promoter in stably transfected cells. A, a
schematic of the mouse SP-C promoter-pBLCAT6 constructs is shown. Hatchedboxes indicate polyadenylation sites. Openboxes represent regions of the murine SP-C
proximal promoter, and +1 indicates the start of
transcription. The plasmid designations are indicated on the right. B, TNF-
inhibited mSP-C CAT and hSP-C
SV40 TAg mRNAs. MLE-15 cells were transfected with each construct, and
two independent pools of stable transformants were treated with 30
ng/ml rmTNF-
for 24 h. Northern blot analysis of 15 µg of RNA
per lane was performed with sequential hybridization to CAT,
SV40 TAg, and L32 probes. Filters were autoradiographed for 4 days, 1
day and overnight, respectively.
Intratracheal administration of TNF- to adult mice
decreased SP-C mRNA in association with mild pulmonary inflammation in vivo. Transcriptional run-on analysis in MLE cells in
vitro demonstrated that the inhibitory effect of TNF-
on SP-C
expression was mediated, at least in part, by decreased SP-C gene
transcription. TNF-
inhibited the activity of both murine and
human SP-C promoter elements in MLE cells, demonstrating that the
effects of TNF-
were determined by cis-active elements located
within 3.7-4.6 kb of the start of transcription. Deletion
analysis of the murine SP-C promoter demonstrated that the inhibitory
activity was mediated by cis-acting elements located within 320 bp of
the start of SP-C gene transcription.
Respiratory distress syndrome
in premature infants and in older infants and adults (ARDS) is
associated with the lack or dysfunction of pulmonary surfactant that
reduces surface tension in the alveolus (see (25) , for
review). Surfactant proteins and phospholipids are both decreased in
ARDS, a condition associated with marked increase in airway
TNF-(1) . The finding that TNF-
inhibited surfactant
protein synthesis and that high levels of TNF-
are associated with
respiratory failure (reviewed in (26) ) supports the concept
that respiratory distress syndrome is caused, at least in part, by
TNF-
-mediated surfactant dysfunction and decreased surfactant
production by alveolar type II cells.
TNF- decreased surfactant
proteins A and B and mRNA in human adenocarcinoma cells in
vitro(27) . In previous studies from this laboratory,
inhibitory effects of TNF-
on human SP-A gene expression were
mediated by inhibition of gene transcription(28) . In contrast,
TNF-
and phorbol 12-myristate 13-acetate inhibited SP-B synthesis
and mRNA by a process dependent on the destabilization of SP-B mRNA
mediated by cis-active elements located in the 3`-untranslated region
of the human SP-B mRNA(8) . The present findings demonstrate
that in addition to the effects of TNF-
on expression of
surfactant proteins A and B, TNF-
markedly inhibits SP-C mRNA and
SP-C gene transcription in vitro. Whether the observed
inhibition of SP-C mRNA in vivo occurs by the same mechanism
remains to be shown.
Like other genes in the respiratory epithelium,
SP-C expression is subject to cell-specific, ontogenetic, and humoral
regulatory influences. SP-C mRNA and transcriptional activity are first
detected in distal respiratory epithelial cells of the developing lung
on days 10-11 of gestation in the mouse(29) .
Lung-specific and developmental control of SP-C expression was
determined by cis-active elements located within 3-4 kb 5` from
the start of transcription in both the mouse and human SP-C
genes(29, 30) . The mouse 4.8 SP-C promoter was
transactivated in HeLa cells by co-expression of thyroid transcription
factor 1 (TTF-1), a nuclear homeodomain containing transcription factor
expressed in distal respiratory epithelial cells in both human and
mouse lung(31) . TTF-1 activates SP-C promoter function by
direct binding to cis-acting elements in the SP-C gene promoter,
located within 320 bp of the start of transcription. ()Recently, DNA binding activity and dimerization of TTF-1
were shown to be reduction/oxidation (redox) regulated in
vitro(32) . Although TNF-
induces oxidative stress,
it remains to be determined whether TNF-
causes changes in TTF-1
binding to the SP-C promoter.
The lack of cell lines expressing SP-C
limited prior analysis of SP-C gene transcription to in vivo studies in transgenic mice bearing SP-C-driven reporter genes. The
recent development of clonal mouse lung epithelial cell lines (MLE-12
and MLE-15) from lung tumors of transgenic mice bearing 3.7SP-C SV40
TAg chimeric gene (13) facilitated the in vitro analysis of SP-C gene transcription. In the present study,
TNF- inhibited promoter activity of both murine and human SP-C
reporter gene constructs in the MLE cell model system. TNF-
was
equally active in the inhibition of the human and murine SP-C promoter
elements consistent with the close sequence conservation of the
5`-flanking region of the SP-C genes in the human and mouse,
particularly within the 320-bp proximal promoter region(17) .
The inhibitory effects of TNF- were observed with recombinant
human and mouse TNF-
and contrasted sharply with the marked
stimulatory effects of human and mouse TNF-
on ICAM-1 gene
expression in vitro and in vivo. The present findings
are consistent with the modulation of SP-C gene transcription by
signaling through the TNF-R1 or the 55-kDa form of the tumor necrosis
factor receptor. Most cell types express one or two distinct receptors
for TNF-
, specifically the type 1 receptor (TNF-R1) or the 55-kDa
form or the type 2 (TNF-R2) 75-kDa form(9, 10) . The
extracellular domains of these receptors contain four conserved
cysteine-rich motifs while the intracellular domains are divergent,
suggesting that the two receptors activate distinct intracellular
signaling pathways. Human TNF-
selectively binds to the mouse
TNF-R1 receptor and does not activate the TNF-R2 receptor(10) .
In the present study, both mouse and human TNF-
inhibited SP-C
gene transcription, supporting the concept that the effects of
TNF-
on SP-C gene expression are mediated by activation of P55 or
type 1 TNF-
receptor. However, the relatively slow time course of
TNF-
inhibition of SP-C expression suggests that changes in SP-C
gene transcription may be mediated by more complex intracellular
signaling pathways than for ICAM-1, a gene known to be activated by P55
receptor signaling(11) . In the present study, TNF-
markedly enhanced ICAM-1 mRNA levels in MLE cells within 8 h after
exposure. TNF-
activates a number of transcription factors
including AP-1, IRF-1 and IRF-2, and NF-IL6 (reviewed in (33) )
and causes the nuclear translocation of transcription factor NF
B
within minutes after TNF-
exposure(34) . TNF-
increased ICAM-1 in fibroblasts and epithelial cells in a process
mediated, at least in part, by translocation of
NF
B(35, 36) . The rapid effects of TNF-
on
ICAM-1 expression in MLE cells were similar to that previously
identified in human bronchial epithelial and pulmonary adenocarcinoma
cells(35, 37) . Inhibitory effects of TNF-
on
SP-C gene transcription in MLE cells occurred more slowly. In human
adenocarcinoma cells, inhibitory effects of TNF-
on SP-A and SP-B
expression were also relatively slow and dependent upon ongoing
transcription(26, 27, 38) . Thus the
mechanisms signaling the inhibitory effects of TNF-
on SP-C
transcription remain unclear at present but appear to be distinct from
those mediating ICAM-1 induction.
TNF--mediated inhibition of
osteocalcin expression in osteoblasts was dependent on TNF-responsive
elements located in the proximal promoter of the human osteocalcin
gene(39) . Comparison of the sequence of the murine SP-C
promoter used in the present study with the consensus sequence for the
TNF-
response element in the human osteocalcin gene (GGC(A/T)GCC)
revealed no obvious homologies. The precise DNA-protein interactions
mediating the effects of TNF-
on SP-C gene expression remain to be
elucidated.
In the present study, TNF- markedly inhibited
surfactant protein C gene expression in association with stimulation of
ICAM-1 mRNA, an adhesion molecule that modulates the binding of
inflammatory cells to the pulmonary epithelium. Enhancement of ICAM-1
expression by TNF-
may exacerbate the recruitment and activation
of leukocytes in the lung, further contributing to lung injury. Thus
the combined effects of TNF-
on surfactant protein and ICAM-1
mRNAs may contribute to the pathogenesis of surfactant dysfunction and
inflammation associated with various forms of lung injury.