(Received for publication, July 24, 1996, and in revised form, November 7, 1996)
From the Departments of Physiology, Surgery, Medicine, and Pathology, Columbia University, College of Physicians and Surgeons, New York, New York 10032
Activation of transcription at the nuclear factor
interleukin 6 (NF-IL-6) DNA binding motif modulates expression of
multiple genes important in host adaptive and developmental mechanisms. Studies showing that hypoxia-induced transcription of IL-6 in cultured
endothelial cells was due to transcriptional activation by the
NF-IL-6 motif in the promoter (Yan, S.-F., Tritto, I., Pinsky, D., Liao, H., Huang, J., Fuller, G., Brett, J., May, L., and Stern, D. (1995) J. Biol. Chem. 270, 11463-11471)
led us to prepare transgenic mice using 115- or 14-base pair regions of the promoter encompassing the NF-IL-6 site ligated to the
lacZ reporter gene and the basal thymidine kinase promoter.
On exposure to hypoxia or induction of ischemia, mice bearing either of
the constructs showed prominent expression of the transgene in lung and
cardiac vasculature and in the kidney but not in the liver (parenchyma
or vasculature). In contrast, transgenic mice bearing a mutationally
inactivated NF-IL-6 site showed no increase in transgene expression in
hypoxia. Gel retardation assays revealed time-dependent,
hypoxia-enhanced nuclear binding activity for the NF-IL-6 site in
nuclear extracts of the heart, lung, and kidney but not in the liver;
the hypoxia-enhanced band disappeared on addition of antibody to
C/EBP-NF-IL-6. Consistent with the specificity of
hypoxia-mediated activation of C/EBP
-NF-IL-6, gel
retardation assays showed no change in the intensity of the
hypoxia-enhanced gel shift band in the presence of excess unlabeled
oligonucleotide probes or antibodies related to other transcription
factors, including NF
B, AP1, cAMP response element-binding protein,
SP1, and hypoxia-inducible factor 1. These data indicate that the
transcription factor NF-IL-6 is sensitive to environmental oxygen
deprivation, and the tissue-specific pattern of gene expression
suggests that local mechanisms have an important regulatory effect.
One of the fundamental cellular responses to environmental stress is redirection of biosynthetic mechanisms that promote adaptation and enhance survival. These mechanisms have been studied in detail following induction of heat shock and are now coming under intensive study in the setting of oxygen deprivation. Limitation of oxygen supply occurs in a range of situations, from environmental deficiency at high altitude to that resulting from diminished blood flow, or in solid tumors with growth that has outpaced the ability of neovasculature to sustain cellular activities. One of the best studied instances of gene expression modulated by hypoxia is induction of erythropoietin (1-3), which appears to be mediated largely at the transcriptional level by hypoxia-inducible factor 1 (HIF-11; Ref. 1). The DNA binding motif for this transcription factor is also present in the promoter of certain key glycolytic enzymes (4) and in a homologous form in the promoter for vascular endothelial growth factor (5-7) and non-insulin-dependent glucose transporter 1 (8), both of which demonstrate enhanced expression in response to oxygen deprivation.
In a previous study, we found that hypoxia stimulated expression of the
cytokine interleukin 6 (IL-6) by cultured endothelial cells (ECs), and
that this was due to enhanced transcription driven by the nuclear
factor IL-6 (NF-IL-6) site in the IL-6 promoter (9). In the hypoxic
cultured endothelium, activation NF-IL-6-C/EBP was observed,
although there was no evidence for activation of C/EBP
or
. Since
NF-IL-6 binding elements are present in multiple genes with expression
that might contribute importantly to the cellular response during
oxygen deprivation (10), we made transgenic mice in which a 115- or
14-bp portion of the IL-6 promoter, encompassing the NF-IL-6 site, was
fused to the lacZ gene as a reporter and to the basal
thymidine kinase promoter. Our results indicate that transcriptional
activation does occur at the NF-IL-6 site in vivo in hypoxia
and ischemia, and that this is especially evident in vasculature of the
lung and heart and in the kidney. In addition, nuclear extracts from
the latter organs showed a prominent hypoxia-mediated increase in
binding activity for an NF-IL-6 oligonucleotide probe. In contrast,
hypoxic liver did not display enhanced transgene expression in
vasculature or parenchyma, and there was no increase in NF-IL-6 binding
activity. These results indicate the existence of tissue-specific
mechanisms underlying activation of NF-IL-6, which can be modulated by
the local microenvironment in response to oxygen deprivation.
Plasmids pTKCi 225/111 (11-13), pYSF52, and pYSF55 (9) were digested with HindIII-BamHI to remove the chloramphenicol acetyltransferase gene. The latter was replaced with the 3.7-kilobase lacZ gene from plasmid pCH110 (14) by ligation with T4 DNA ligase in one orientation to produce plasmids pYSF46, pYSF56, and pYSF57 from pTKCi 225/111, pYSF52, and pYSF55, respectively (9). The JM109 strain of Escherichia coli was used for heat shock transformation of the plasmid constructs.
Generation of Transgenic Mice and DNA AnalysisA
3.9-kilobase DNA segment was released from pYSF46, and a 3.8-kilobase
DNA segment was released from pYSF56 and pYSF57 by ApaI-BamHI double digests. The first linearized
DNA fragment from pYSF46 contained the indicated portion (225 to
111) of the IL-6 promoter (9, 11-13) and is termed construct 1 (Fig.
1A). The second fragment, from pYSF56 (termed construct 2),
contained the 14-bp NF-IL-6 site (
158 to
145) and has previously
been shown to regulate IL-6 expression in cultured endothelial cells
exposed to hypoxia (9). The third fragment, from pYSF57, termed
construct 3, contained a 4-bp mutation previously shown to inactivate
the NF-IL-6 site (9, 11-13). In addition, all constructs contained the
thymidine kinase basal promoter (
80 to +18; Refs. 12 and 14),
lacZ, and a polyadenylation signal from SV40. The excised fragments were visualized by ethidium bromide staining of agarose gels
(0.8%), and purified according to the manufacturer's instructions (QIAEX II; Qiagen, Chatsworth, CA). DNA was diluted to 5 ng/µl in
Tris-HCl (7.5 mM) and EDTA (0.2 mM, pH
7.4).
Superovulated female mice (B6CBAF1/J; Jackson Laboratory, Bar Harbor, ME) were mated with B6CBAF1/J males, and fertilized eggs were harvested and microinjected with the appropriate construct. In brief, following incubation for 18 h at 37 °C in M16 media (Specialty Media, Lavallette, NJ) in an atmosphere with carbon dioxide (5%) in air, oocytes were transferred to the oviducts of pseudopregnant foster mothers. Three independent transgenic lines for construct 1, four for construct 2, and two for the mutant construct (construct 3) were generated. To identify transgenic mice, the tails (~1 cm) were cut from 3-week-old pups, and DNA was extracted and digested with EcoRI-HindIII. Southern blotting was performed using a 32P-labeled EcoRI-HindIII fragment from pCH110. Founders were than mated with nontransgenic mice to generate new offspring (B6CBAF1/J) and to check transmission of the transgene.
Studies in Hypoxic MiceAll experiments were performed according to protocols approved by the Institutional Care and Use Committee at Columbia University, in accordance with Association for the Accreditation of Laboratory Animal Care guidelines. Mice were subjected to hypoxia using a custom designed controlled environmental chamber as described previously (15). Animals were placed in the chamber and allowed free access to food and water. During the first hour, the oxygen content of the atmosphere in the chamber was slowly reduced from ambient levels to 6% with the balance of the gas mixture made up of nitrogen. Then, 6% oxygen content was maintained for the following 6 h. Mice were then sacrificed, and tissue (lung, heart, kidney, and liver) was harvested for the assays described below. For the mouse lung ischemia model, after appropriate anesthesia, mice were placed on a Harvard ventilator (tidal volume, 0.75 ml; respiratory rate, 180/min), ventilated with 100% oxygen, and underwent bilateral thoracotomy. The left pulmonary artery was cross-clamped for a period of 60 min, and the cross-clamp was then released. After 4 h of reperfusion, lung tissue from the nonmanipulated (nonischemic) and manipulated (postischemic) mice was obtained and either placed in formalin (10%) for histologic analysis or snap frozen in liquid nitrogen.
Assays for Transgene ExpressionELISA to evaluate
expression of -galactosidase used tissue fragments (lung, heart,
kidney, and liver) from normoxic or hypoxic transgenic mice washed
three times with phosphate-buffered saline. Organs were then suspended
in homogenizing buffer (NaCl, 100 mM; Tris-HCl, pH 7.4, 20 mM; EDTA, 1 mM; aprotinin, 1 µg/ml;
phenylmethylsulfonyl fluoride, 1 mM) and homogenized using
a Polytron. Soluble protein was isolated, and its concentration was
determined using the Bio-Rad protein assay kit. Equal amounts of total
protein were analyzed in each ELISA for
-galactosidase (Life
Technologies, Inc.).
Immunohistological analysis of -galactosidase expression was studied
in the lung, heart, kidney, and liver. Organs were rapidly harvested
from normoxic or hypoxic animals, fixed overnight in formalin (3.5%),
dehydrated, and embedded in paraffin by standard procedures (16).
Sections were then rehydrated, incubated in blocking buffer
(phosphate-buffered saline containing bovine serum albumin, 1%, and
normal goat serum, 2%) for 30 min at 37 °C, washed in
phosphate-buffered saline, and exposed to monospecific rabbit anti-
-galactosidase IgG (30 µg/ml; Cortex Biochem Inc., San
Leandro, CA). Tissue sections were washed again in phosphate-buffered
saline and incubated with biotinylated goat anti-rabbit immunoglobulin followed by peroxidase-conjugated ExtrAvidin (Sigma).
Localization of peroxidase conjugates was revealed using
aminoethylcarbazole as the chromogen. Nonimmune rabbit IgG replaced the
anti-
-galactosidase IgG for controls. IL-6 was localized in tissue
from normoxic or hypoxic transgenic mice using affinity-purified
anti-mouse IL-6 IgG and the same immunostaining procedure as described
previously (9). This antibody was generously provided by Dr. Gerald
Fuller (University of Alabama, Birmingham, AL).
Nuclear
extracts were prepared from organs of nontransgenic mice (B6CBAF1/J),
including lung, heart, kidney, and liver (mice had been exposed to
normoxic or hypoxic conditions for 0.5, 1, 2, 3, and 4 h) using
the method of Dignam et al. (17). These extracts were
prepared in an environment with the same ambient oxygen tension as that
in the experiment. Complementary 14-bp oligonucleotides (158 to
145) containing an NF-IL-6 site that included 5
-ACATTGCACAATCT-3
and 5
-AGATTGTGCAATGT-3
were used. Oligonucleotides were annealed
and 5
-end-labeled with [32P]ATP (3000 Ci/mmol) using T4
polynucleotide kinase according to standard procedures. Binding
reactions were performed by preincubation of nuclear extract protein
(1-5 µg) in HEPES (pH 7.9, 20 mM), KCl (60 mM), MgCl2 (1 mM), EDTA (0.1 M), glycerol (10%), dithiothreitol (0.5 mM),
and poly(dI-dC) (2 µg) at room temperature for 10 min followed by
addition of the double-stranded 32P-labeled oligonucleotide
(~35 fmol) and a second incubation at room temperature for 20 min.
Where indicated, antiserum to C/EBP
,
, or
, CRP1, CREB-1,
CREB-2, c-Fos, c-Jun/AP1, p50 (NF
B), or p65 (NF
B) (Santa Cruz
Biotechnology, Santa Cruz CA) was incubated with nuclear extract at
room temperature for 1 h, and then the procedure described above
was followed. Samples (5 µg of protein in each lane) were loaded
directly onto nondenaturing polyacrylamide-bisacrylamide gels (4%)
prepared in Tris borate/EDTA (0.5 ×; Tris, 45 mM, boric acid, 45 mM, and EDTA, 0.1 M), and
electrophoresis was performed at room temperature for 1.5-2 h at 200 V. For competition studies, a 100-fold molar excess of unlabeled
NF-IL-6 probe (as above), SP1 probe (18), 18-bp probe for HIF-1 (1),
CREB (this and the other the following oligonucleotides probes were
purchased from Santa Cruz), AP1, and NF
B was used.
Having previously demonstrated
that hypoxia-mediated induction of IL-6 in cultured endothelial cells
was due to activation of transcription at the C/EBP-NF-IL-6 site in
the promoter (9), we generated transgenic mice containing this DNA
binding motif. The bacterial gene lacZ and the basal
thymidine kinase promoter were placed under transcriptional control of
the 5
-regulatory sequence of the IL-6 gene from positions
225 to
111 and
158 to
145 (with respect to the transcription start site
at +1) (Fig. 1A, constructs 1 and
2). The segment of the promoter
225 to
111 has been
previously shown to function as an enhancer in response to hypoxia in
cultured endothelial cells because of the presence of the
C/EBP
-NF-IL-6 motif at
158 to
145. Additional transgenic mice
were made in which the C/EBP
-NF-IL-6 motif was mutationally inactivated (Fig. 1A, construct 3). To create transgenic
mice, the indicated construct was isolated, linearized, and
microinjected into fertilized mouse eggs. Three and four different
founders were generated with constructs 1 and 2, respectively, each of which transmitted the transgene. Southern analysis of these lines is
shown in Fig. 1B (constructs 1, lanes 2-4, and
2, lanes 5-8). Two founders were identified with the
mutationally inactivated C/EBP
-NF-IL-6 construct 3 (Fig. 1B,
lanes 9 and 10). Note the negative control in Fig.
1B, lanes 11 and 12, in which the same amount of
DNA from nontransgenic mice was loaded and the positive control (Fig.
1B, C), in which the EcoRI-HindIII DNA
fragment from pCH110 was used as the sample.
Transgenic mice bearing either
construct 1 or 2 were subjected to hypoxia in a controlled environment
chamber in which the oxygen concentration was held constant at about
6%. Under these conditions, mice displayed normal activity and food
consumption over the experimental period, although they were visibly
tachypneic, indicative of hypoxia. First, we studied expression of the
transgene in mice bearing construct 1 in the lung, heart, kidney, and
liver. ELISA demonstrated 4-6-fold increased -galactosidase antigen in the lung, heart, and kidney following exposure of animals to hypoxia
versus controls maintained in normoxia (Fig.
2). The heart showed the greatest increase in
-galactosidase, about 6-fold. Immunostaining for
-galactosidase
demonstrated transgene expression in hypoxic pulmonary vasculature
(Fig. 3A), endothelium (Fig. 3A)
and especially smooth muscle cells (the latter depicted at high
magnification; Fig. 3A, inset), a distribution similar to our previously reported hypoxia-induced expression of IL-6 (9). In
normoxic controls, staining for
-galactosidase antigen was virtually
undetectable in the lung (Fig. 3B), analogous to the low
levels of IL-6 antigen noted before (9). In the heart, immunostaining
for
-galactosidase was observed in hypoxic coronary vasculature and
in myocardium (Fig. 3C); there was no staining in normoxic
controls (Fig. 3D). To be certain that this pattern of
transgene expression indicated expression of a gene regulated by
NF-IL-6-C/EBP
, we also looked for IL-6 antigen in hypoxic cardiac
tissue. Immunostaining demonstrated that IL-6 was expressed in hypoxic
cardiac vasculature and myocardium (Fig. 3E) but not in
normoxic controls (Fig. 3F). In the kidney, antigen was also virtually absent in normoxic controls (Fig. 3H), whereas
after hypoxia there was staining in the vasculature and proximal
tubules (Fig. 3G); this distribution of
-galactosidase
staining was also comparable with that observed using antibody to IL-6
(Fig. 3I). There was only minimal staining for IL-6 in
normoxic mouse kidney (Fig. 3J).
In contrast to the results in the lung, heart, and kidney, the livers
of transgenic mice bearing construct 1 showed no significant induction
of -galactosidase antigen by ELISA (Fig. 2) or by immunostaining (Fig. 4, A and B show hypoxic and
normoxic liver, respectively). Consistent with these findings, IL-6
transcripts (Fig. 4C) were not increased in livers of mice
subjected to hypoxia (lanes N for normoxia and H
for hypoxia) compared with the induction previously demonstrated
in the lung (9) and also the enhancement observed in heart and kidney
(data not shown). In contrast, infusion of mice with lipopolysaccharide
resulted in induction of IL-6 transcripts in the liver (Fig. 4C,
lane LPS for lipopolysaccharide infusion), as reported previously
(19).
Since the region of 225 to
111 used to prepare transgenic mice with
construct 1 contains multiple possible regulatory elements, we also
made mice in which only the NF-IL-6 site from the IL-6 promoter was
present (
158 to
145), i.e. construct 2 (Fig. 1B, lanes 5-8). When exposed to hypoxia, these animals demonstrated enhanced expression of
-galactosidase in a similar pattern and to a
degree similar to that observed with mice bearing construct 1 (data not
shown). Importantly, mice bearing construct 3, in which the NF-IL-6 was
mutationally inactivated, showed no increase in
-galactosidase
expression compared with normoxic controls in the lung or other organs
(Fig. 5, C and D, hypoxic and
normoxic lung, respectively). This contrasts with increased levels of
-galactosidase antigen in lungs from hypoxic mice with the wild-type
NF-IL-6 site in mice bearing either construct 1 (115 bp; Fig.
4A) or construct 2 (14 bp); Fig. 5 shows
-galactosidase
staining in hypoxic versus normoxic lung with construct 2 (Fig. 5, A and B, respectively). In addition,
mice with construct 3 subjected to hypoxia did not show transgene
expression in the lung (Fig. 5, C and D), the
heart (Fig. 5, E and F), or the kidney (Fig. 5,
G and H, hypoxic and normoxic tissues,
respectively).
Response of Transgenic Mice to Ischemia
To extrapolate
our hypoxia model to the setting of organ ischemia, a method for
inducing ischemia of the left lung was developed, and activation of the
transgene and expression of IL-6 were assessed. Using transgenic mice
bearing constructs 1 and 2, increased -galactosidase expression was
observed by immunocytochemistry in ischemic lung vasculature (Fig. 6,
A and C, respectively), but there
was no expression of the transgene in the nonmanipulated right lung
(Fig. 6, B and D). Detection of IL-6 antigen by
immunostaining also showed a similar vascular pattern of enhanced
expression in the ischemic left lung compared with the nonmanipulated
right lung (Fig. 6, E and F, respectively).
Response of Mice to Hypoxia: Activation of C/EBP
Expression of IL-6 and the two transgenes
containing the NF-IL-6 site was inducible by hypoxia in a
tissue-specific manner. Whereas vascular endothelium and smooth muscle
in the lung demonstrated high levels of transgene expression on
exposure to hypoxia, neither hepatocytes nor hepatic vasculature showed
significantly enhanced -galactosidase expression. To probe the
nature of hypoxia-mediated transcriptional activation, EMSA was
performed using nuclear extracts from the lung, heart, kidney, and
liver. Nuclear binding activity for the 32P-labeled NF-IL-6
probe from the IL-6 promoter (
158 to
145) (11-13) showed a strong
increase in the lung, heart, and kidney (Fig.
7A). In the lung, the intensity of the gel
shift band increased within 2 h of hypoxia, was sustained up to
4 h, and was due to sequence-specific binding to the NF-IL-6
oligonucleotide probe, as shown by competition with excess unlabeled
probe for NF-IL-6 but not with unlabeled probes for NF
B, AP1, CREB,
SP1, or HIF-1 (Fig. 7B). Supershift experiments confirmed
the involvement of C/EBP
-NF-IL-6; addition of antibody to
C/EBP
to the reaction mixture resulted in dose-dependent inhibition
of the gel shift band (Fig. 7C). At IgG dilutions of
1:10-1:1000, appearance of the gel shift band was
completely blocked, whereas at higher dilutions, 1:5000-1:10,000, the band was again observed. In
contrast to these results with C/EBP
, antibody to C/EBP
or
or
CRP1 had no effect on migration of the gel shift band (Fig.
7C). Studies with antibodies to other transcription factor
components, including c-Fos, c-Jun/AP1, CREB-1, CREB-2, p50 (NF
B)
and p65 (NF
B), did not change the intensity or migration of the gel
shift band (data not shown). The hypoxia-enhanced gel shift band
binding to the NF-IL-6 oligonucleotide probe with hypoxic lung nuclear
extracts was qualitatively and quantitatively different from the band
observed with extracts from normoxic lung. In addition to the gel shift
band being of much lower intensity in the normoxic tissue (compared
with hypoxia), there was no supershift change in intensity of the band
with antibody to C/EBP
,
, or
, CRP1, CREB-1, CREB-2, p65
(NF
B), p50 (NF
B), c-Jun/AP1, and c-Fos (data not shown).
To further assess the nature of components binding to the NF-IL-6 probe
in nuclear extracts of hypoxic kidney, competition and supershift
studies were performed. The intensity of the hypoxia-enhanceable gel
shift band in nuclear extracts of kidney was greatly diminished in the
presence of excess unlabeled NF-IL-6 but not by excess unlabeled
NFB, AP1, CREB, SP1, or HIF-1 (data not shown). Supershift experiments with nuclear extracts from hypoxic kidney also indicated a
pattern similar to what was observed with hypoxic lung; anti-C/EBP
antibody completely blocked the appearance of the band (at a 1:10 dilution of antibody, the gel shift band is gone, but at a 1:5000 dilution, the band is again seen), whereas antibody to c-Fos, c-Jun/AP1, CREB-1, CREB-2, p65, or p50 had no effect (data not shown).
Hypoxic stress accompanies a range of pathophysiologically relevant situations. Modulation of gene expression in response to oxygen deprivation is a basic component of adaptation to such an environmental perturbation (20-23). We previously noted induction of IL-6 in cultured ECs subjected to hypoxia, which we speculated might have a protective role by suppressing the effect of potent proinflammatory mediators, such as tumor necrosis factor (9, 24-25), elicited by hypoxemia and/or ischemia. IL-6 induction in hypoxic cultured ECs was driven by increased gene transcription mediated by the NF-IL-6 site in the promoter (9). Mice subjected to hypoxia also demonstrated increased vascular expression of IL-6, corresponding with our observations in vitro. These results led us to examine whether hypoxia would trigger NF-IL-6-mediated transcription in vivo.
To address this issue, two types of transgenic mice were
prepared: mice bearing a transgene spanning a 115- and 14-bp portion of
the IL-6 promoter, in each case constituting the NF-IL-6 site. Expression of both transgenes was observed in lung vasculature, as
expected from our previous results (9). In addition, expression of the
-galactosidase reporter was evident in the heart, in both cardiac
myocytes and vasculature, and in the kidney, especially in proximal
tubule cells. In contrast, the liver showed no significant hypoxia-mediated increase in transgene expression, either in
hepatocytes or in the vasculature. These data suggested the existence
of mechanisms for organ-specific regulation of hypoxia-inducible gene
expression mediated by NF-IL-6, analogous to cell-specific expression
of the erythropoietin gene in transgenic mice (although expression of
the NF-IL-6 transgene follows a distinct pattern; Ref. 26). In this
context, EMSA from hypoxic lung, heart, and kidney showed striking
increases in nuclear binding activity for the NF-IL-6 oligonucleotide
probe, whereas there was no increase in hypoxic liver. Pilot
experiments with cultured hepG2 cells (data not shown) and our past
studies on cultured ECs (9) confirmed these differences in the cellular
response to oxygen deprivation. ECs exhibited a rapid and pronounced
increase in NF-IL-6 binding activity in response to hypoxia, whereas
this did not occur in hepG2 cells. These data contrast with the well
known hypoxia-mediated activation of HIF-1 in hepG2 cells (8) and
suggest the existence of distinct pathways leading to expression of
transcription factor-DNA binding activity.
Our findings provide a first step in understanding the contribution of
NF-IL-6 motifs to the regulation of gene expression in hypoxia and
ischemia. C/EBP-NF-IL-6-driven transcription was observed in
vasculature of the heart, lung, and kidney and in cardiac myocytes. In
contrast, no expression was observed in the liver, either in
parenchymal cells or vasculature. This suggests the importance of local
factors in the microenvironment in regulation of gene expression.
Future work will be directed toward identification of such autocrine
and paracrine factors produced by hypoxic and ischemic tissues.
We gratefully acknowledge the assistance of Dr. Gerald Fuller, Lester May (New York Medical College), and Dr. Gabriel Godman (Columbia University).