Ceramide decreases surfactant protein B gene expression via down regulation of
TTF-1 DNA binding activity
Loretta Sparkman, Hemakumar Chandru and Vijayakumar Boggaram
Department of Molecular Biology
University of Texas Health Center at Tyler
Tyler, TX 75708-3154
Running title: Ceramide regulation of SP-B gene expression
Address correspondence to:
Vijay Boggaram, Ph. D.
Department of Molecular Biology
11937 US Highway 271
Tyler, TX 75708-3154
Phone: (903) 877-7780
Articles in PresS. Am J Physiol Lung Cell Mol Physiol (September 23, 2005). doi:10.1152/ajplung.00275.2005
Copyright © 2005 by the American Physiological Society.
Ceramide, a sphingolipid, is an important signaling molecule in the inflammatory response.
Mediators of acute lung injury such as TNF-α, PAF and Fas/Apo ligand stimulate sphingomyelin
hydrolysis to increase intracellular ceramide levels. Surfactant protein B (SP-B), a hydrophobic
protein of pulmonary surfactant, is essential for surfactant function and lung stability. In this
study we investigated the effects of ceramide on SP-B gene expression in H441 lung epithelial
cells. Ceramide decreased SP-B mRNA levels in control and dexamethasone treated cells after
24 h incubation and inhibition of SP-B mRNA was associated with inhibition of immunoreactive
SP-B. In transient transfections assays, ceramide inhibited SP-B promoter activity indicating that
the inhibitory effects are exerted at the transcriptional level. Deletion mapping experiments
showed that the ceramide responsive region is located within –233/-80 bp region of human SP-B
promoter. Electrophoretic mobility shift and reporter assays showed that ceramide reduced the
DNA binding activity and transactivation capability of thyroid transcription factor 1 (TTF-
1/Nkx2.1), a key factor for SP-B promoter activity. Collectively these data showed that ceramide
inhibits SP-B gene expression by reducing the DNA biding activity of TTF-1/Nkx2.1
transcription factor. Protein kinase C inhibitor bisindolylmaleimide and the protein tyrosine
kinase inhibitor genistein partially reversed ceramide inhibition indicating that protein kinases
play important roles in the ceramide inhibition of SP-B gene expression. Chemical inhibitors of
de novo ceramide synthesis and sphingomyelin hydrolysis had no effect on TNF-α inhibition of
SP-B promoter activity and mRNA levels suggesting that ceramide does not play a role in the
Key words: sphingolipids; lung injury, inflammation; transcription
Surfactant, a proteolipid complex is essential for lung stability. Surfactant protein B (SP-B) is
an 8 kDa hydrophobic protein of surfactant which promotes the formation and stability of the
surfactant monolayer on the alveolar surface through its interactions with dipalmitoyl
phosphatidylcholine, the principal surface-active phospholipid of surfactant (10). SP-B gene
expression is subject to developmental and multifactorial regulation by cytokines, growth factors
and hormones (5). SP-B mRNA is expressed in a cell/tissue-restricted manner by the alveolar
type II and Clara cells of the lung.
SP-B is essential for lung function. A complete lack of SP-B as in the case of frame-shift
mutation in codon 121 (121ins2) in humans (25), and in genetically engineered SP-B null mice
(9) results in death due to respiratory failure. Partial deficiency of SP-B is associated with
susceptibility to lung injury suggesting that optimal SP-B levels are necessary for maintenance of
lung function (8). Apart from newborn respiratory distress syndrome (RDS) and congenital
alveolar proteinosis, SP-B levels are also reduced in a variety of lung diseases such as acute
respiratory distress syndrome (ARDS) (13), respiratory syncitial virus (RSV) infection in infants
(15), familial interstitial lung disease (2) and others. Inflammatory mediators such as TNF-α (26)
(4) and nitric oxide (30) inhibit SP-B gene expression.
The sphingolipid ceramide serves as an important signaling molecule in the inflammatory
response triggered by a host of stress factors including cytokines, ionizing radiation and others
(22). Ceramide can be produced via de novo synthesis or hydrolysis of membrane sphigomyelin
by sphingomyelinases. Lung cells express high levels of sphingomyelinases (29, 35), and the
levels of lactosylceramide, a ceramide derivative, and ceramide are markedly elevated in the
bronchoalveolar lavage fluid of ARDS patients (27) and in plasma of sepsis patients respectively
(11). In sepsis, plasma ceramide levels are correlated with mortality (11). A role for ceramide in
the development of lung injury is indicated by increased lung permeability and surfactant
dysfunction in rats exposed to TNF-α and ceramide (29) and suppression of platelet activating
factor induced lung edema in acid sphingomyelinase deficient rats (12). These data strongly
suggest a role for ceramide in the development of acute lung injury. A potential mechanism for
ceramide induced lung injury is by inhibiting surfactant protein gene expression. In particular
inhibition of SP-B can result in lung injury as reduced SP-B levels are correlated with surfactant
dysfunction and susceptibility to lung injury. In this study we tested the proposal that high
concentrations of ceramide decrease SP-B mRNA levels in lung epithelial cells. To date no
information is available on the sphingolipid regulation of surfactant protein gene expression.
We found that ceramide inhibited basal and dexamethasone induced SP-B mRNA levels in
H441 lung epithelial cells. Ceramide inhibition of SP-B mRNA was associated with inhibition
of SP-B promoter activity and ceramide response region was mapped between –233/-80 bp of
SP-B promoter. Ceramide inhibition of SP-B mRNA was associated with inhibition of DNA
binding activity of TTF-1. Pharmacological inhibition of protein kinase C partly reversed
ceramide inhibition indicating a role for the protein kinase C signaling pathway.
Cell culture. NCI-H441 cells [American Type Culture Collection (ATCC) HTB-174], a
human lung adenocarcinoma cell line with characteristics of bronchiolar (Clara) epithelial cells
were grown on plastic tissue culture dishes in RPMI 1640 medium containing 10% fetal bovine
serum, penicillin (100u/ml), streptomycin (100 µg/ml) and amphotericin B (0.25 µg/ml). In all
experiments the medium was changed to RPMI 1640 without serum for 24 h before the start of
Materials. C2-ceramide, sphingosine and sphingosine-1-phosphate, were obtained from
Avanti (Alabaster, AL). Dihydroceramide C2 was from Sigma. C2-ceramide, dihydroceramide
C2 and sphingosine were dissolved in anhydrous ethanol. Sphingosine-1-phosphate was
dissolved in a mixture of methanol:water (95:5) at a concentration of 0.5 mg/ml by heating at
45oC for 10-15 min followed by sonication for 10 secs each time for 3 times. Solubilized
sphingosine-1-phosphate was dried under nitrogen and reconstituted in cell culture medium
containing 0.4% bovine serum albumin. TNF-α was purchased from R & D sytems
(Minneapolis, MN). Bisindolylmaleimide, genistein, PP2 were from Calbiochem (La Jolla, CA).
Dimethylaminopurine was from Acros Organics. TRI Reagent was from Molecular Research
Center (Cincinnati, OH).
RNA isolation and Northern blot analysis. Experimental procedures for RNA isolation and
Northern blot analysis are as described previously (21). Total RNA was isolated by the acid-
guanidinium thiocyanate-phenol method using TRI Reagent (Molecular Research Center,
Cincinnati, OH). RNA was quantified by measuring absorbance at 260 nm, and equal amounts of
RNA were separated by electrophoresis on agarose gels (1%) containing 20 mM MOPS and
1.1% formaldehyde. Separated RNAs were transferred to HybondN+ membrane by capillary
action with saline sodium citrate (SSC) (20x) as the transfer solution. The membranes were UV
cross-linked, hybridized to 32P-labeled human SP-B and GAPDH cDNAs and washed. The final
wash was routinely done with 1 x SSC containing 0.1% SDS at 65oC. The washed blots were
scanned with a PhosphorImager and RNA bands corresponding to SP-B and GAPDH were
quantified. SP-B mRNA levels were normalized to 18S rRNA levels to correct for variations in
the quantification, loading and transfer of RNA. The expression of GAPDH mRNA was assessed
as an internal control.
Immunohistochemical detection of SP-B. Immunoreactive SP-B was detected with a staining
kit from Lab Vision (Fremont, CA) according to the manufacturer’s protocol. H441 cells grown
on cell culture slide chambers were fixed in Excell PLUS for 1 h and endogenous peroxidase
activity blocked by incubation with Peroxide Block for 30 min. Afterward, cells were incubated
with rabbit polyclonal human SP-B antibodies (1:200 dilution; Chemicon International,
Temecula, CA) followed by horse radish peroxidase conjugated secondary antibody. Cells were
then incubated with aminoethylcarbazole to visualize the antigen-antibody complex and
counterstained with contrast blue solution.
Transient transfection and reporter gene assay. Amplification of human SP-B 5’ flanking
DNA containing –911/+41 bp and construction of luciferase reporter plasmid have been reported
previously (30). 5’ truncated DNA fragments containing –517/+41, -233/+41 and –80/+41 bp
SP-B promoter were amplified by PCR with a plasmid containing –911/+41 bp of SP-B 5’
flanking DNA as the template and the following SP-B primers containing introduced Sac I and
Hind III sites (underlined),
5’-CGAGCTCCATGTGTCCATAGAACCAGA-3’ (-517/-498) (sense)
5’-CGAGCTCAGCCACAAGTCCAGGAACAT-3’ (-233/-214) (sense)
5’-CGAGCTCACTGAGGTCGCTGCCACTCC-3’ (-80/-61) (sense)
Amplified DNAs were ligated upstream of the luciferase reporter gene in the plasmid
pGL3luc(basic) (Promega, Madison, WI). SP-B promoter plasmids were sequenced to ensure
that they are free of nucleotide changes. TTF-1 and HNF-3 reporter plasmids containing multiple
copies of SP-B TTF-1 and HNF-3 binding sites linked upstream of basal SP-B promoter
(-59/+41 bp) were constructed by PCR. A plasmid containing –236/+41 bp of SP-B 5’ flanking
DNA served as the template and the following SP-B primers were used,
TTF-1: 5’-CGAGCTCA G C A C C T G G A GGGCTCTTCAGAGCAAGCACCTGGAGGG
CTCTTCAGAGCACTACAGAGCCCCCACGCCCCGCCCAGCT-3’ (-59/-32) (sense)
CCCCGCCCAGCT-3’ (-59/-32) (sense)
Sac I and Hind III sites introduced into the primers are underlined. Introduced TTF-1 and HNF-3
sequences are shown in italics and core TTF-1 and HNF-3 binding sites are underlined.
Amplified DNA was inserted upstream of luciferase reporter gene in the vector pGL3basic and
sequenced to ensure that it did not contain any mutations. Plasmid DNAs were amplified in
Escherichia coli Top10 strain (Invitrogen) and purified by anion exchange chromatography
(Qiagen). DNAs were transiently transfected into cells by liposome-mediated DNA transfer with
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were
cotransfected with a β-galactosidase expression plasmid, pcDNA3.1 (Invitrogen) for the
assessment of transfection efficiency. After transfection, cells were first incubated overnight in
serum-containing medium and then in serum-free medium for 24 h after which they were
subjected to treatments. β-galactosidase and luciferase reporter activities in cell extracts were
measured by chemiluminiscent assays (Tropix, Bedford, MA and Promega, Madison, WI).
Preparation of nuclear extracts and electrophoretic mobility shift analysis (EMSA). Nuclear
extracts were prepared according to the methods described previously (31) (32). Protein
concentrations of nuclear extracts were determined by Bradford’s method using Bio-Rad protein
Synthetic oligonucleotides were annealed by heating equimolar concentrations of sense and
antisense oligonucelotides in 10 mM Tris-HCl, pH 7.5 containing 10 mM MgCl2 and 50 mM
NaCl at 95oC for 5 min and then allowed to cool to room temperature over a period of 1 h. The
concentration of annealed oligonucleotides was determined by measuring absorbance at 260 nm
(50 ng/ml = 1.0 A260 unit). The sense strand sequences of SP-B promoter oligonucleotides are as
TTF-1/Nkx2.1: 5’-GCACCTGGAGGGCTCTTCAGAGCAA-3’ (-111/-87)
HNF-3: 5’-GCAAAGACAAACACTGAG-3’ (-90/-73)
Double stranded oligonucleotides were 5’end labeled using [γ32P] and T4 polynucleotide
kinase. EMSAs were performed essentially as described previously (19) by incubating 0.5-1.0 ng
(100,000 cpm) of the labeled oligonucleotide with 5 µg of nuclear protein in 20 µl of binding
buffer [13 mM Hepes, pH 7.9 containing 13% glycerol, 80 mM KCl, 5 mM MgCl2, 1 mM DTT,
1 mM EDTA and 1 µg of poly (dI-dC) as non-specific competitor DNA] at 30oC for 20 min. For
antibody supershift assay, protein-DNA complex was first formed and then incubated with
T/EBP antiserum or antibody for 20 min at room temperature. Polyclonal antisera to the N-
terminal portion of rat T/EBP (TTF-1/Nkx2.1) was kindly supplied by Dr. Shioko Kimura,
National Cancer Institute, Bethesda, MD. After electrophoresis, the gel was dried and exposed
to an X-ray film.
Statistics. Data are shown as means ± SD/SE. In experiments where SP-B mRNA levels in
control cells were arbitrarily set at 100%, statistical significance was analyzed by one-sample t-
test. For other samples, unpaired t-test was used to analyze statistical significance. One-tailed P
values of <0.05 were considered significant.
Ceramide inhibits SP-B mRNA and promoter activity. No information is available about
sphingolipid regulation of surfactant protein gene expression. To begin to ascertain the role of
sphingolipid metabolites in the regulation of surfactant protein gene expression, we first
determined the effects of ceramide on the expression of SP-B mRNA. We studied the effects of
ceramide on the basal and dexamethasone induction of SP-B mRNA levels to determine if
ceramide has similar effects on SP-B levels in the absence and presence of dexamethasone.
Also, as SP-B mRNA levels are significantly increased in dexamethasone treated cells the effects
of dexamethsone and other agents on SP-B mRNA can be readily assessed. H441 cells were
incubated with a cell permeable C2-ceramide analog with or without dexamethasone and its
effects on SP-B mRNA levels determined by Northern blotting. We found that ceramide
inhibited basal (control = 100, ceramide = 58 ± 8.7, n = 4) and dexamethasone induction (control
= 100, dex = 1409 ± 372, n = 4, dex + ceramide = 430 ± 104, n = 4) of SP-B mRNA levels after
24 h of incubation without significant effects on GAPDH mRNA levels (Fig. 1). The inhibitory
effects of ceramide were time-dependent, and significant effects were observed after 24 h of
incubation (Fig. 2). Consistent with its inhibitory effects on SP-B mRNA levels, ceramide
reduced SP-B promoter activity by similar extent (control = 100, ceramide = 48 ± 3.1, n = 3)
indicating that the inhibitory effects are exerted at the transcriptional level (Fig. 2). Ceramide at a
concentration of 10 µM did not have significant toxic effects on H441 cells as judged by light
microscopy, total RNA yield and GAPDH mRNA expression levels. Ceramide at a concentration
of 10 µM was used in all the experiments.
Ceramide decreases SP-B protein levels. We analyzed SP-B protein levels by
immunohistochemical detection to determine if ceramide inhibition of SP-B mRNA levels is
associated with inhibition of SP-B protein. H441 cells grown on cell culture slide chambers were
incubated in control medium or dexamethasone (10-7 M) containing medium with or without
ceramide (10 µM) for 24 h and SP-B levels were visualized by immunohistochemical detection.
Results (Fig. 3) showed that SP-B levels were induced by treatment with dexamethasone and
incubation with ceramide reduced SP-B levels to control levels. Although immunohistochemical
detection does not provide quantitative data, these results showed that ceramide inhibited SP-B
Sphingosine and sphingosine-1-phosphate do not inhibit SP-B mRNA levels. Ceramide once
produced via hydrolysis of membrane sphingomyelin or de novo synthesis is further metabolized
into sphingosine and sphingosine-1-phosphate by ceramidase and sphingosine kinase enzymes
respectively. Sphingosine and sphingosine-1-phosphate have diverse effects on cells including
effects on gene expression (33). We assessed the effects of these sphingomyelin metabolites on
SP-B mRNA levels. As SP-B mRNA levels are rather low in H441 cells, in the following
experiments we determined the effects of different agents and inhibitors on dexamethasone
induction of SP-B mRNA levels. Our earlier data showed that ceramide had similar inhibitory
effects on SP-B mRNA levels in control and dexamethasone treated cells. We found that
sphingosine (10 µM) and sphingosine-1-phosphate (1 µM) did not inhibit SP-B mRNA levels
indicating that the inhibitory effects of ceramide are selective (Fig. 4). Higher concentrations of
sphingosine-1-phosphate (up to 5 µM) had no effect on SP-B mRNA levels (data not shown).
Further dihydroceramide an inactive analog of ceramide was significantly less effective than
ceramide to inhibit SP-B mRNA levels. Consistent with the lack of effects on SP-B mRNA
levels, dihydroceramide, sphingosine and sphingosine-1-phosphate did not inhibit SP-B
promoter activity (data not shown).
Ceramide response region is located between –236 and –80 bp of SP-B promoter. Our
experiments showed that ceramide inhibited SP-B mRNA levels by inhibiting SP-B promoter
activity. We mapped SP-B promoter region responsible for ceramide inhibition by deletion
analysis. We found that deletion of SP-B 5’ flanking DNA from –991 bp to –233 bp had no
effect on ceramide inhibition of SP-B promoter activity, however further deletion to –80 bp
rendered the promoter insensitive to inhibition indicating that the SP-B promoter region between
–236 and –80 bp contains DNA elements necessary for inhibition (Fig. 5). Deletion of 5’ region
of SP-B flanking DNA resulted in a gradual loss of the promoter activity with the –80/+41 bp
fragment retaining 1-5 % of the activity of the –911/+41 bp fragment.
Ceramide decreases TTF-1/Nkx2.1 DNA binding activity. Deletion mapping analysis of SP-B
5’ flanking DNA showed that the ceramide response region is located within –233/-80 bp of
promoter region. This region that is part of the SP-B promoter contains functionally important
binding sites for HNF-3 and TTF-1 transcription factors (6) (20). As this region contains HNF-3
and TTF-1 DNA binding sites it is possible that ceramide inhibition of SP-B promoter activity is
mediated via down regulation of DNA binding activities of these transcription factors. We
determined the effect of ceramide on the DNA binding activities of TTF-1 and HNF-3 elements
by EMSA. HNF-3 DNA binding activity in H441 cells is mainly due to HNF-3α as there is no
detectable expression of HNF-3β and HNF-3γ (7). Treatment of cells with ceramide (10 µM)
resulted in decreased TTF-1 DNA binding activity in a time-dependent manner with significant
effects after 24 h of incubation (Fig. 6). In contrast the DNA binding activity of HNF-3 was not
affected (Fig. 6). The inactive ceramide analog, dihydroceramide, did not reduce TTF-1 DNA
binding activity (data not shown) indicating that the inhibitory effects of ceramide are specific.
In separate experiments we found that the non-immune IgG/serum did not produce super-shifted
bands with TTF-1 probe and the TTF-1 antiserum/antibodies produced super-shift bands only in
A549 cells transfected with a TTF-1 expresssion plasmid but not in untransfected cells showing
the specificity of the TTF-1 antibody. A549 cells have either very low or undetectable levels of
TTF-1. We further assessed the role of TTF-1 and HNF-3 transcription factors in the ceramide
inhibition of SP-B promoter activity by determining the effects of ceramide on the transcriptional
activities of TTF-1 and HNF-3 reporter plasmids. Reporter constructs containing multiple TTF-1
and HNF-3 binding elements placed upstream of basal SP-B promoter were transfected into
H441 cells and the effect of ceramide on reporter activity was determined. Results (Fig. 7)
showed that ceramide inhibited TTF-1 but not HNF-3 reporter expression indicating that
ceramide negatively regulates TTF-1 binding activity resulting in reduced transactivation
capability. The significance of an increase in HNF-3 reporter activity in ceramide treated cells is
not clear. Taken together these data strongly indicated that ceramide down regulates TTF-1 DNA
binding to inhibit SP-B promoter activity.
Role of protein kinase signaling pathways in the ceramide inhibition of SP-B mRNA.
Many of the effects of cytokines, growth factors and bioactive lipids on cell growth,
differentiation and gene expression are mediated via protein kinase signaling pathways. To gain
insights into signaling pathways and underlying mechanisms that mediate ceramide inhibition of
SP-B mRNA, we investigated the effects of chemical inhibitors of various protein kinases on the
ceramide inhibition of SP-B mRNA. Our preliminary results showed that ceramide did not
activate ERK, p38 and JNK MAPK signaling pathways (Chandru, H and Boggaram, V.,
unpublished observations) indicating that these pathways may not be necessary for ceramide
inhibition. Inhibition by ceramide was unaffected by the nitric oxide synthase inhibitor, L-
NAME, indicating that nitric oxide does not mediate inhibition of SP-B mRNA levels
(Sparkman, L and Boggaram, V., unpublished observations). We determined the effects of
pretreatment with bisindolylmaleimide I (protein kinase C inhibitor), PP2 (Src kinase inhibitor),
genistein (protein tyrosine kinase inhibitor) and dimethylaminopurine (ceramide activated
protein kinase inhibitor) on ceramide inhibition of dexamethsone induction of SP-B mRNA.
Results (Fig. 8) showed that bisindolylmaleimide I and genistein significantly blocked the
inhibitory effects of ceramide. An other protein kinase C inhibitor, Go6983, also partly reversed
ceramide inhibition of SP-B mRNA levels (data not shown) indicating that protein kinase C
signaling may be involved in ceramide inhibition. Src kinase inhibitor PP2 synergized
dexamethasone induction of SP-B mRNA and appeared to block ceramide inhibition. Analysis of
the effect of ceramide on the activation of Src kinase by western blotting produced negative
results suggesting that Src kinase signaling pathway may not be involved (data not shown).
Further studies are necessary to clarify the role of PP2 and the Src kinase pathway in the
ceramide inhibition of SP-B mRNA. The inability of dimethylaminopurine to block ceramide
inhibition indicated that ceramide activated protein kinase signaling pathway may not be
necessary for ceramide inhibition.
TNF-α inhibition of SP-B promoter activity and mRNA levels is not mediated via changes in
ceramide levels. TNF-α is a proinflammatory cytokine whose levels are elevated in a number of
inflammatory diseases of the lung including ARDS. Activation of sphingomyelinases is an
important pathway leading to intracellular increases in ceramide levels, and TNF-α is a known
activator of sphingomyelinases in rat lung (29) and H441 cells (35). TNF-α inhibits SP-B mRNA
levels in mouse lung (26) and H441 cells (4) by post-transcriptional (mRNA stability) and
transcriptional mechanisms respectively. Considering that TNF-α activates sphingomyelinases to
increase ceramide levels in the lung, we sought to understand the role of ceramide in the TNF-α
inhibition of SP-B promoter activity and mRNA levels. De novo ceramide synthesis was blocked
with myriocin (14), a specific inhibitor of serine palmitoyl transferase and sphingomyelin
hydrolysis was blocked with imipramine (18) and 3-O-methylsphingomyelin (17), inhibitors of
acid and neutral sphingomyelinases respectively, and the effects of TNF-α on the inhibition of
SP-B promoter activity and SP-B mRNA levels were determined. Results (Fig. 9) showed that
consistent with previous findings TNF-α inhibited SP-B promoter activity and SP-B mRNA
levels and inhibition of ceramide synthesis and sphingomyelin hydrolysis had no effect on TNF-
α inhibition. Imipramine and 3-O-methyl sphingomyelin showed modest inhibitiory effects on
SP-B promoter activity and mRNA levels while myriocin had modest stimulatory effects. These
data indicated that TNF-α inhibition of SP-B promoter activity and mRNA levels is not mediated
via changes in the intracellular ceramide levels.
Mediators of acute lung injury such as TNF-α, PAF and LPS activate sphingomyelinases to
increase intracellular ceramide levels. Ceramide has long been recognized as a signaling
molecule in the inflammatory response. As surfactant serves important roles in lung stability any
alterations in surfactant composition/or function can lead to surfactant dysfunction and
susceptibility to lung injury. Recently it was shown that TNF-α acting via ceramide (29), and
sphingosine (28) decreased surfactant function and the activity of CTP:phosphocholine
cytidyltransferase (35), a rate-limiting enzyme in surfactant phospholipid synthesis, respectively
suggesting that surfactant is a key target for the development of inflammation induced lung
Our data showed that ceramide reduced basal and dexamethasone induced SP-B mRNA and
protein levels in H441 cells. The inhibition was specific for ceramide as the inactive analog
dihydroceramide was significantly less effective to repress SP-B mRNA. Metabolites of
ceramide, such as sphingosine and sphingosine-1-phosphate did not inhibit SP-B mRNA levels
indicating the specificity of ceramide inhibition. Our studies used the semi-synthetic and cell
permeable C2-ceramide analog. It is not clear if the inhibitory effects are due to ceramide itself
or mediated by metabolites of ceramide such as ceramide-1-phosphate. Ceramide reduced SP-B
promoter activity indicating that transcriptional mechanisms play important roles in the
repression of SP-B gene expression. To our knowledge repression of SP-B gene expression by a
bioactive lipid has not been reported previously. The effect of ceramide on the expression of
other surfactant protein genes has not yet been investigated. Our preliminary findings showed
that ceramide at 10 and 20 µM inhibited SP-A mRNA levels after 24 h of incubation in H441
cells. Our data showing inhibitory effects of ceramide on SP-B gene expression provides a
molecular basis for ceramide mediated lung injury as in the case of ARDS wherein elevated
ceramide levels are detected in the bronchoalveolar lavage fluid (27). There is limited
information on ceramide regulation of gene expression in the lung. Ceramide induces COX-2
expression in A549 lung epithelial cells independently of NF-kB activation (24). Ceramide
inhibition of CTP: phosphocholine cytidyltransferase synthesis was attributed to multiple
signaling pathways including protein kinase C, p38 MAPK and cytosolic phospholipase A2 (3).
Molecular mechanisms of ceramide regulation of gene expression and associated signaling
pathways in the lung remain to be elucidated.
Deletion mapping analysis identified SP-B promoter region between –233/-80 bp to contain
cis-DNA elements necessary for ceramide inhibition. This promoter region contains HNF-3 and
TTF-1 sites that are necessary for promoter activity. Ceramide treatment of H441 cells resulted
in decreased DNA binding activity of TTF-1/Nkx2.1 and decreased reporter activity from TTF-1
reporter plasmid while the DNA binding and reporter activity of HNF-3 were not affected.
Collectively these data suggested that ceramide inhibition of TTF/Nkx2.1 DNA binding activity
leads to inhibition of SP-B mRNA levels. SP-B promoter function is sensitive to changes in the
helical phasing and orientation of cis-DNA elements (1) and is dependent on the combinatorial
interactions between TTF-1, HNF-3 and Sp1/Sp3 transcription factors (20). It is likely that
ceramide inhibition of TTF-1/Nkx2.1 DNA binding activity interferes with the assembly of the
transcriptional complex resulting in the inhibition of SP-B transcription. Our data showed that
ceramide inhibited basal and dexamethasone induced SP-B mRNA levels. Our data also showed
that ceramide inhibited SP-B promoter activity indicating that transcription plays important roles
in the inhibition. The inductive effects of dexamethasone are primarily due to enhanced
stabilization of SP-B mRNA (21) suggesting that ceramide reduces SP-B mRNA levels in
dexamethasone treated cells by inhibiting transcription. Whether ceramide has any effect on the
stability of SP-B mRNA in dexamethasone treated cells remains to be determined.
TTF-1/Nkx2.1 is a lung and thyroid restricted homeodomain containing trancription factor
that is a key activator of surfactant protein gene expression (23). Molecular mechanisms of
ceramide inhibition of TTF-1/Nkx2.1 DNA binding activity remain to be determined. TTF-
1/Nkx2.1 DNA binding activity is sensitive to changes in the phosphorylation (37) (36) and
redox (34) status. As such ceramide induced changes in the phosphorylation and/or redox status
of TTF-1 could contribute to reduced DNA binding activity. It is unlikely that ceramide induced
changes in TTF-1 redox status are responsible for the reduced TTF-1 DNA binding activity as a
number of antioxidants such as, n-acetylcysteine, reduced glutathione, mannitol,
dimethylsulfoxide and dimethylthiourea failed to reverse inhibition of SP-B mRNA (data not
shown). Whether ceramide causes changes in the phosphorylation status of TTF-1 to inhibit its
DNA binding activity remains to be investigated. Alternatively ceramide could decrease TTF-1
expression to reduce its DNA binding activity. TNF-α inhibition of SP-B mRNA levels in H441
lung cells has been attributed to reduced TTF-1 binding activity (4).
Our preliminary experiments showed that ceramide did not activate ERK, p38 and JNK
MAPK and Src-kinase signaling pathways in H441 cells (data not shown) indicating that
ceramide inhibition of SP-B gene expression may be dependent on other signaling pathways.
Partial reversal of ceramide inhibition by protein kinase C inhibitors suggests a possible role for
protein kinase C signaling in the ceramide inhibition of SP-B gene expression.
TNF-α, a proinflammatory cytokine is a key mediator of acute lung injury and TNF-α
inhibition of surfactant synthesis is a contributing factor for lung injury (16). Our data showed
that chemical inhibitors of de novo ceramide synthesis and sphigomyelin hydrolysis did not
block TNF-α inhibition of SP-B promoter activity and mRNA levels indicating that ceramide
does not mediate TNF-α inhibition. Other mediators of acute lung injury such as platelet
activating factor (PAF) and lipopolysaccharide (LPS) also activate sphingomyelinases to elevate
intracellular ceramide levels. It remains to be determined if PAF and LPS inhibit SP-B gene
expression via increases in the intracellular ceramide levels. Ceramide repression of SP-B gene
expression provides a molecular basis for surfactant dysfunction in inflammatory diseases such
This work has been supported by the National Heart, Lung and Blood Institute grant
HL48048. We thank James B. McKnight for technical assistance and Dr. Barry Starcher and G.
Koteswara Rao for help with immunohistochemical detection of SP-B.
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Fig. 1. Ceramide inhibits SP-B mRNA levels in H441 lung epithelial cells. H441 cells were
incubated in control medium (C) or medium containing dexamethasone (10-7 M) (Dex)
± ceramide (Cer.) at indicated concentrations for 24 h and SP-B and GAPDH mRNAs
were analyzed by Northern blotting. A. A representative Northern blot showing the
effects of treatments on SP-B and GAPDH mRNA levels. 18S rRNA levels detected by
ethidium bromide staining are also shown. B. SP-B levels in control cells were arbitrarily
set at 100 and the levels in treated cells were determined relative to control levels. Data
represent mean ± SEM of 4 independent experiments. **P < 0.01 for ceramide (10 and
20 µM)-treated cells vs control cells. *P <0.05 for dexamethasone-treated cells vs control
cells. **P < 0.01 for dex + ceramide (10 µM) and dex + ceramide (20 µM)-treated cells
vs dexamethasone alone-treated cells.
Fig. 2. Ceramide inhibits SP-B mRNA levels and SP-B promoter activity in a time-
dependent manner in H441 cells. A. Cells were incubated in control medium (C) or
medium containing ceramide (Cer.) (10 µM) for the indicated periods of time and SP-B
mRNA levels were determined. Data shown are means ± SEM of 3 independent
experiments. #P < 0.0001 for ceramide-treated cells vs control at 24 h. B. Cells
transfected with a plasmid containing –911/+41 bp of human SP-B 5’ flanking DNA
linked to luciferase reporter gene in the vector pGL3luc (basic) were incubated in control
medium (C) or medium containing ceramide (10 µM) for the indicated periods of time.
Luciferase activity in cell lysates was determined and normalized to cotransfected β-
galactosidase activity. Data represent means ± SEM of 3 independent experiments. *P <
0.05 for ceramide-treated cells vs control at 12 h and **P <0.01 for ceramide-treated cells
vs control at 24 h.
Fig. 3. Ceramide inhibits immunoreactive SP-B protein levels in H441 cells. Cells were
incubated in control medium, medium plus dexamethasone (10-7 M) or medium
containing dexamethasone (10-7 M) plus ceramide (10 µM) for 24 h and then processed
for immuohistochemical detection of SP-B as described in the methods section. Arrows
indicate staining for SP-B immunoreactive protein. Results shown are representative of 2
Fig. 4. Sphingolipid inhibition of SP-B mRNA levels in H441 cells is specific for ceramide.
Cells were incubated in control medium (C) or medium containing dexamethasone (Dex)
(10-7 M) ± ceramide (Cer.) (10 µM), dihydroceramide (10 µM), sphingosine (10 µM) or
sphingosine-1-phosphate (S-1-P) (1 µM) for 24 h and SP-B and GAPDH mRNA levels
were analyzed by Northern blotting. Sphingosine-1-phosphate was reconstituted in
medium containing 0.4% bovine serum albumin. A. A representative Northern blot
showing the effects of treatments on SP-B and GAPDH mRNA levels. 18S rRNA levels
visualized by ethidium bromide staining are also shown. B. Data shown are means ±
SEM of 5 independent experiments. #P < 0.0001 for dexamethasone-treated cells vs
control and for dexamethasone + ceramide-treated cells vs dexamethasone alone-treated
cells. *P < 0.05 for dexamethasone + dihydroceramide-treated cells vs dexamethasone
Fig. 5. Deletion mapping identifies SP-B promoter region –233/-80 bp to contain ceramide
response DNA elements. SP-B-luciferase promoter plasmids containing 5’ deletions
were transiently transfected into H441 cells and transfected cells were incubated in
control medium (C) or medium containing ceramide (Cer.) (10 µM) for 24 h. Luciferase
activity in cell lysates was determined and normalized to cotransfected β-galactosidase
activity. A. Schematic diagram of SP-B 5’ flanking DNA showing the locations of
functionally important DNA elements and the transcription start site. B. Data shown are
means ± SEM of 4 independent experiments. **P <0.01 for ceramide-treated cells vs
control for the –911/+41 and –233/+41 bp constructs. #P < 0.0001 for ceramide-treated
cells vs control for the –517/+41 bp construct.
Fig. 6. Ceramide reduces TTF-1 DNA binding activity in H441 cells. H441 cells were
incubated in control medium or medium containing ceramide (10 µM) for the indicated
periods of time and nuclear extracts were prepared. TTF-1 and HNF-3 DNA binding
activities were analyzed by antibody-supershift EMSA and EMSA respectively. Dotted
and solid arrows indicate the mobilities of antibody-protein-DNA and protein-DNA
complexes. Similar results were obtained in 3 other independent experiments.
Fig. 7. Ceramide reduces TTF-1 reporter activity in H441 cells. A. A schematic diagram of
the TTF-1 and HNF-3 reporter plasmids. The reporter plasmids contain 4 copies of TTF-
1 or HNF-3 binding sequences inserted upstream of –59/+41 bp fragment of human SP-B
gene in the pGL3luc(basic) vector. B. H441 cells were transiently transfected with TTF-1
and HNF-3 reporter plasmids along with pcDNA3.1, a β-galactosidase expression
plasmid and then incubated in control medium (C) or medium containing ceramide (Cer.)
(10 µM) for 24 h. Luciferase activities in cell lysates were determined and normalized to
cotransfected β-galactosidase activity. Data shown are means ± SEM of 5 independent
experiments. ***P < 0.001 for ceramide treated cells vs control for TTF-1 reporter
plasmid. **P < 0.01 for ceramide treated cells vs control for HNF-3 reporter plasmid.
Fig. 8. Effects of protein kinase inhibitors on ceramide inhibition of SP-B mRNA levels.
H441 cells were first incubated for 2 h in medium containing inhibitors and then
incubation continued with dexamethasone (10-7 M) ± ceramide (10 µM) for 24 h. SP-B
mRNA levels were analyzed by Northern blotting and normalized to 18S rRNA levels. C,
control; Dex, dexamethasone; BIM, bisindolylmaleimide I (5 µM); PP2, 4-amino-5-(4-
chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (10 µM); Gen., genistein (50 µM);
DMAP, dimethylaminopurine (1 mM). Data are means ± SEM of 4 independent
experiments. **P < 0.01 for dex vs control, *P <0.05 for Dex + PP2 vs Dex alone, **P <
0.01 for Dex + Ceramide vs Dex alone, *P < 0.05 for BIM + Dex + Ceramide vs Dex +
Ceramide, P = 0.052 for Genistein + Dex + Ceramide vs Dex + Ceramide.
Fig. 9. Effects of sphingomyelinase and ceramide synthesis inhibitors on TNF-α α inhibition
of SP-B promoter activity and SP-B mRNA levels. A. H441 cells were transiently
transfected with a plasmid containing –911/+41 bp of human SP-B 5’ flanking DNA
linked to luciferase reporter gene in the vector pGL3luc(basic). Transfected cells were
first incubated ± imipramine (25 µM), 3-O-methylsphingomyelin (MSM) (10 µM) or
myriocin (1 µM) for 2 h and then incubation continued ± TNF-α (25 ng/ml) for an
additional 24 h. Luciferase activity in cell lysates was determined and normalized to
cotransfected β-galactosidase activity. Data shown are means ± SD of 2 independent
experiments. **P < 0.01 for TNF-α vs control; P = 0.3 for imipramine + TNF-α vs TNF-
α alone; P = 0.29 for 3-O-methylsphingomyelin + TNF-α vs TNF-α alone; P = 0.22 for
myriocin + TNF-α vs TNF-α alone. B. H441 cells were first incubated ± imipramine (25
µM), 3-O-methylsphingomyelin (MSM) (10 µM) or myriocin (1 µM) for 2 h and then
incubation continued ± TNF-α for an additional 24 h. SP-B mRNA levels were analyzed
by Northern botting and normalized to 18 S rRNA levels. Data shown are means ± SD of
2 independent experiments. *P < 0.05 for TNF-α vs control; P = 0.15 for imipramine +
TNF-α vs TNF-α alone; P = 0.14 for 3-O-methylsphingomyelin + TNF-α vs TNF-α
alone; P = 0.22 for myriocin + TNF-α vs TNF-α alone.
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