©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tumor Necrosis Factor- Inhibits Surfactant Protein C Gene Transcription (*)

(Received for publication, December 14, 1994; and in revised form, April 28, 1995)

Cindy J. Bachurski Gloria S. Pryhuber (§) Stephan W. Glasser Susan E. Kelly Jeffrey A. Whitsett (¶)

From the Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio 45229-3039

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha (TNF-alpha), decreased SP-C mRNA within 24 h of intratracheal administration to mice. In vitro, TNF-alpha 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-alpha 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-alpha. TNF-alpha 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-alpha 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-alpha may contribute to the abnormalities of surfactant homeostasis associated with pulmonary injury and infection.


INTRODUCTION

Acute respiratory distress syndrome (ARDS) (^1)is a common, often fatal complication of infection, shock, and trauma and has been associated with increased levels of the proinflammatory cytokine, TNF-alpha(1) . TNF-alpha 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-alpha causes increased pulmonary ICAM-1 expression, acute lung inflammation, and pulmonary edema(5, 6, 7) . In vitro, TNF-alpha 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-alpha on SP-C gene expression have not been previously reported.

In the lung, TNF-alpha 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-alpha, 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-alpha 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-alpha 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-alpha signaling in various lung cell types.

In the present study, TNF-alpha 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-alpha on SP-C gene expression in respiratory epithelial cells.


MATERIALS AND METHODS

Intratracheal Administration of TNF-alpha in the Mouse

Two- to three-month-old, pathogen-free, FVB/N mice were anesthetized with methoxyflurane vapor. Intratracheal inoculation was performed by exposing the trachea with an anterior midline incision and insertion of a 27-gauge needle attached to a tuberculin syringe parallel to the trachea. A 50-µl inoculum was dispensed into the lungs followed by approximately 0.2 cc of air. The incision was closed with 1 drop of Nexaband liquid (Veterinary Products Laboratories, Phoenix, AZ). At 24 h after administration, mice were sacrificed by exsanguination after lethal intraperitoneal injection of sodium pentobarbital (Nembutal, Abbott). Total RNA was isolated from the left lung and, in some experiments, from both left and right lower lobes with similar results.

Cell Culture and Transfection

Murine lung epithelial cell lines MLE-12 and MLE-15 were maintained in HITES medium with 2% fetal bovine serum as described(13) . MLE cells are clonal lines derived from pulmonary tumors in mice transgenic for the human 3.7 SP-C SV40 large T antigen chimeric gene(14) . These lines share several characteristics with pulmonary type II cells including production of surfactant proteins and phospholipids and the presence of multilamellar bodies and microvilli(13) . Cells were used at passages 10-20 in culture. Recombinant human TNF-alpha (rhTNF-alpha) and mouse TNF-alpha (rmTNF-alpha) were kindly provided by Genentech (Palo Alto, CA). Additional rhTNF-alpha was purchased from Boehringer Mannheim. At the time of treatment with TNF-alpha, cells were maintained in HITES medium with or without hydrocortisone with similar results. At the indicated time points, monolayers were washed with Hanks' balanced salt solution (Life Technologies, Inc.) and lysed in 4 M guanidinium thiocyanate, 25 mM Hepes, pH 7, 0.5% Sarkosyl, 0.1 M 2-mercaptoethanol(15) . Total RNA was isolated using phase lock gels (5 Prime 3 Prime, Gaithersburg, MD).

MLE-15 cells were co-transfected with CAT reporter constructs (described below) and pRSVneo using the CaPO(4) 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.

Reporter Constructs

Murine SP-C promoter sequences were retrieved as several large overlapping Lambda Dash clones from a murine 129/J genomic library (gift of Roger Askew, Department of Molecular Genetics, University of Cincinnati) using previously published (17) human SP-C cDNA sequences as probes. A 5-kb XbaI fragment was excised and inserted into pUC19's multiple cloning site at XbaI and consists of 4.8-kb promoter sequences, the first exon, and a portion of the first intron. The intron and coding portion of the first exon were removed by Bal-31 digestion from a unique KpnI site, blunt end ligated to XhoI linkers, and liberated by an XhoI and AccI digest. This fragment was directionally cloned into pBLCAT 6 (18) using the same enzymes. Sequencing determined that this construct (p4.8 muCAT) contains a 4.8-kb murine promoter sequence and 18 bp of the first exon followed by the introduced XhoI site and then the vector-derived reporter gene, CAT. Deletions of p4.8 muCAT were produced using restriction enzymes, which cut uniquely in both the murine promoter and in the multiple cloning region of pBLCAT6 (SphI for p0.8 muCAT and PstI for the p0.32 muCAT).

S1 Nuclease Analysis and Northern Blotting

S1 nuclease mapping was done essentially as described previously(19) . Linearized probes were end-labeled with [-P]ATP, combined, and hybridized overnight at 56 °C with total lung RNA (2 µg) or MLE cell RNA (10 µg). Protected fragments were liberated by digestion with 110 units of S1 nuclease in the presence of excess unlabeled carrier DNA for 1 h at room temperature. The fragments were resolved on 8 M urea, 6% polyacrylamide gels, visualized by autoradiography, and quantitated using a Molecular Dynamics PhosphorImager.

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 beta-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 beta-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-alpha treatment caused a slight induction of beta-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 alpha-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.

Statistical Analysis

PhosphorImager data from Northern and S1 nuclease protection assays of mouse lung RNA were analyzed by single-factor analysis of variance and Fischer's protected least significant difference statistic, utilizing Statview 4.0 statistical analysis software (Abacus Concepts, Berkeley, CA) on a Macintosh computer system. The data were normalized to L32 and are presented as a fraction of the mRNA detected in saline-treated lungs.

Northern Blot and Run-on Probes

The CAT GenBlock (Pharmacia Biotech Inc.) was random primer labeled with [alpha-P]dCTP and used as a probe for CAT mRNA. Clone K41.1, containing the mouse ICAM-1 cDNA (23) , was digested with EcoRI and HindIII, and the 1.39-kb fragment encompassing the 3` half of the cDNA was used as a probe for Northern blot analysis. For cytoplasmic beta-actin, the 5` half of the murine actin cDNA(21) , Asp718 to BglII, was used for Northern blots and subcloned into the Asp718 to BamHI sites of M13 mp18 as a run-on probe. A 1.1-kb HindIII fragment of the SV40 genome (map units 4002-5171) encompassing a portion of the coding region of the SV40 large and small T antigen genes was used for Northern blots and was subcloned into M13 mp18 for use as a run-on probe. An Asp718 to BamHI fragment of the mouse SP-C gene spanning exons 3-6 was subcloned into M13 mp19 for run-on analysis. Identity and orientation of the run-on probes were confirmed by dideoxy sequence analysis.

Nuclear Run-on Assays

Preparation of nuclei and nuclear run-on assays were done essentially as described previously(24) . Briefly, nuclei were harvested from approximately 5 10^7 cells by gentle vortexing in 700 µl of ice-cold lysis buffer (10 mM NaCl, 3 mM MgCl(2), 10 mM Tris-HCl at pH 7.4, 5 mM dithiothreitol, 0.5% Nonidet P-40). Nuclei were pelleted by centrifugation, and cytoplasmic RNA was isolated from the supernatant by adding 2 volumes of guanidinium thiocyanate buffer and processing as for whole cell RNA (see above). The nuclei were resuspended in an equal volume of storage buffer (40% glycerol, 50 mM Tris-HCl at pH 8, 5 mM MgCl(2), 0.1 mM EDTA) and frozen at -80 °C until assayed.

To label nascent transcripts, nuclei were thawed on ice and incubated in 25 mM HEPES (pH 7.4), 2.5 mM MgCl(2), 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 [alpha-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 alpha-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 10^7 cpm/ml.


RESULTS

TNF-alpha Decreases SP-C mRNA in Vivo

Intratracheal administration of TNF-alpha to adult mice inhibited SP-C mRNA within 24 h (Fig. 1). In contrast, ICAM-1 mRNA was markedly induced by TNF-alpha while MRPL32 (L32), a ribosomal protein mRNA, was unaltered. The animals appeared well and continued normal activities after TNF-alpha administration. Changes in SP-C mRNA occurred in association with histologic evidence of mild pulmonary inflammation, monocytic infiltration, and decreased SP-B mRNA content. (^2)SP-A mRNA levels were not significantly altered by TNF-alpha treatment (data not shown). Both rhTNF-alpha and rmTNF-alpha significantly decreased SP-C mRNA and increased ICAM-1 mRNA in vivo.


Figure 1: SP-C and ICAM-1 mRNA after intratracheal administration of murine or human TNF-alpha. Adult FVB/N mice were treated with a single dose of 50 µg of human or mouse TNF-alpha 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-alpha-treated groups are indicated:**, p < 0.0001; *, p = 0.01; #, p = 0.05 (Fisher's protected least significant difference statistic).



TNF-alpha Inhibits SP-C mRNA in MLE Cells in Vitro

MLE-12 cells, a murine pulmonary epithelial cell line expressing SV40 large T antigen (TAg) under control of the human 3.7SP-C gene promoter(13) , were used as a model system to study the mechanisms by which TNF-alpha inhibits SP-C mRNA. Exposure of MLE-12 cells to mouse or human TNF-alpha decreased endogenous SP-C mRNA 3-5-fold as assessed by S1 nuclease protection analysis (Fig. 2, toppanel). The endogenous ICAM-1 mRNA and the SV40 TAg mRNA, produced from the human 3.7SP-C SV40 TAg construct used to immortalize the cells, were measured by Northern blot analysis. Human and mouse TNF-alpha markedly decreased SV40 TAg mRNA and increased ICAM-1 mRNA (Fig. 2, bottompanel).


Figure 2: Murine and human TNF-alpha inhibit SP-C mRNA and induce ICAM-1 mRNA in vitro. MLE-12 cells were treated with human or mouse TNF-alpha 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-alpha, lane4 with 60 ng/ml rmTNF-alpha, lane5 with 30 ng/ml rhTNF-alpha, and lane6 with 60 ng/ml rhTNF-alpha. 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-alpha 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-alpha 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-alpha. TNF-alpha 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-alpha significantly inhibited SP-C mRNA in vitro.


Figure 3: TNF-alpha inhibits endogenous SP-C and transgenic human 3.7SP-C SV40 TAg mRNAs in vitro. MLE12 cells were treated with increasing concentrations of rmTNF-alpha for 24 h, and total RNA was isolated. Upperpanels represent S1 nuclease protection assays for SP-C and beta-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 beta-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-alpha on SP-C mRNA. MLE-12 cells were treated with 25 ng/ml rmTNF-alpha, 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 beta-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, beta-actin, and ICAM-1 mRNAs. Data representative of three experiments are shown.



Transcriptional Run-on Analysis

TNF-alpha inhibited transcription of the endogenous mouse SP-C and human 3.7SP-C promoter-driven SV40 TAg genes in MLE-12 cells as assessed by nuclear run-on assay (Fig. 5A). The inhibitory effect of TNF-alpha on gene transcription increased with longer incubation times and was maximal by 16 h of exposure to TNF-alpha (Fig. 5B). Transcription of beta-actin was unaffected by TNF-alpha treatment. The observed inhibition of SP-C and SV40 TAg gene transcription was similar in extent and timing to the decrease in steady state mRNA levels.


Figure 5: TNF-alpha 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-alpha. Nuclei were harvested, and nascent transcripts were elongated in the presence of [alpha-P]UTP. 2 10^7 dpm/ml were hybridized to slot blots of single-stranded probes for beta-actin, SV40 TAg (TAg), murine SP-C, and M13 mp19 vector DNA. alpha-Amanatin (alphaAM, 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(0) = 1.0.



Deletion Analysis of Murine SP-C Promoter

Constructs containing 4.8-0.32 kb of the murine SP-C proximal promoter were sufficient to confer inhibitory effects of TNF-alpha on reporter gene expression. Portions of the murine SP-C promoter were fused to the reporter gene, CAT, and stably transfected into MLE-15 cells (constructs are diagrammed in Fig. 6A). TNF-alpha decreased CAT mRNA in independently generated pools of stably transfected cells harboring the murine SP-C promoter deletion-CAT constructs, including constructs containing 320 bp 5` of the start of SP-C gene transcription (Fig. 6B).


Figure 6: TNF-alpha 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-alpha 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-alpha 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.




DISCUSSION

Intratracheal administration of TNF-alpha 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-alpha on SP-C expression was mediated, at least in part, by decreased SP-C gene transcription. TNF-alpha inhibited the activity of both murine and human SP-C promoter elements in MLE cells, demonstrating that the effects of TNF-alpha 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-alpha(1) . The finding that TNF-alpha inhibited surfactant protein synthesis and that high levels of TNF-alpha are associated with respiratory failure (reviewed in (26) ) supports the concept that respiratory distress syndrome is caused, at least in part, by TNF-alpha-mediated surfactant dysfunction and decreased surfactant production by alveolar type II cells.

TNF-alpha 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-alpha on human SP-A gene expression were mediated by inhibition of gene transcription(28) . In contrast, TNF-alpha 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-alpha on expression of surfactant proteins A and B, TNF-alpha 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. (^3)Recently, DNA binding activity and dimerization of TTF-1 were shown to be reduction/oxidation (redox) regulated in vitro(32) . Although TNF-alpha induces oxidative stress, it remains to be determined whether TNF-alpha 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-alpha inhibited promoter activity of both murine and human SP-C reporter gene constructs in the MLE cell model system. TNF-alpha 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-alpha were observed with recombinant human and mouse TNF-alpha and contrasted sharply with the marked stimulatory effects of human and mouse TNF-alpha 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-alpha, 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-alpha 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-alpha inhibited SP-C gene transcription, supporting the concept that the effects of TNF-alpha on SP-C gene expression are mediated by activation of P55 or type 1 TNF-alpha receptor. However, the relatively slow time course of TNF-alpha 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-alpha markedly enhanced ICAM-1 mRNA levels in MLE cells within 8 h after exposure. TNF-alpha 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 NFkappaB within minutes after TNF-alpha exposure(34) . TNF-alpha increased ICAM-1 in fibroblasts and epithelial cells in a process mediated, at least in part, by translocation of NFkappaB(35, 36) . The rapid effects of TNF-alpha 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-alpha on SP-C gene transcription in MLE cells occurred more slowly. In human adenocarcinoma cells, inhibitory effects of TNF-alpha 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-alpha on SP-C transcription remain unclear at present but appear to be distinct from those mediating ICAM-1 induction.

TNF-alpha-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-alpha 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-alpha on SP-C gene expression remain to be elucidated.

In the present study, TNF-alpha 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-alpha may exacerbate the recruitment and activation of leukocytes in the lung, further contributing to lung injury. Thus the combined effects of TNF-alpha on surfactant protein and ICAM-1 mRNAs may contribute to the pathogenesis of surfactant dysfunction and inflammation associated with various forms of lung injury.


FOOTNOTES

*
This work was supported by Program in Molecular Biology Heart and Lung Grant HL40043, the Cystic Fibrosis Foundation, Training in Perinatal Biology Grant NRSA HD07200 (to C. J. B.), Training in Pulmonary and Cardiovascular Development Grant HL07752 (to S. E. K.), and National Institutes of Health Grant HL50046 (to S. W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pediatrics, Strong Children's Medical Center, Rochester, NY 14642.

To whom correspondence should be addressed: Children's Hospital Medical Center, Division of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-559-4830; Fax: 513-559-7868.

(^1)
The abbreviations used are: ARDS, adult respiratory distress syndrome; TNF-alpha, tumor necrosis factor alpha; ICAM-1, intercellular adhesion molecule 1; rmTNF-alpha, recombinant mouse TNF-alpha; rhTNF-alpha, recombinant human TNF-alpha; TNF-R1, TNF-alpha receptor type 1; TNF-R2, TNF-alpha receptor type 2; SP-A, SP-B, and SP-C, surfactant proteins A, B, and C; SV40 TAg, simian virus 40 large tumor antigen; CAT, chloramphenicol acetyltransferase; MLE, murine lung epithelial; bp, base pair(s); kb, kilobase pair(s); TTF-1, thyroid transcription factor 1.

(^2)
G. S. Pryhuber, unpublished observations.

(^3)
S. E. Kelly, C. J. Bachurski, and S. W. Glasser, unpublished observations.


ACKNOWLEDGEMENTS

We thank K. Heminger for excellent technical assistance and Ann Maher for preparation of the manuscript.


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