(Received for publication, June 20, 1995)
From the
We reported recently that the gene that encodes tyrosine
hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of
catecholamines, is regulated by hypoxia in the dopaminergic cells of
the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A.,
Lawson, E. E. & Millhorn, D. E.(1992) J. Neurochem. 58,
1538-1546) and in pheochromocytoma (PC12) cells (CzyzykKrzeska,
M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E.(1994) J.
Biol. Chem. 269, 760-764). Regulation of this gene during
low O conditions occurs at both the level of transcription
and RNA stability. Increased transcription during hypoxia is regulated
by a region of the proximal promoter that extends from -284 to
+27 bases, relative to transcription start site. The present study
was undertaken to further characterize the sequences that confer
O
responsiveness of the TH gene and to identify
hypoxia-induced protein interactions with these sequences. Results from
chloramphenicol acetyltransferase assays identified a region between
bases -284 and -150 that contains the essential sequences
for O
regulation. This region contains a number of
regulatory elements including AP1, AP2, and HIF-1. Gel shift assays
revealed enhanced protein interactions at the AP1 and HIF-1 elements of
the native gene. Further investigations using supershift and
shift-Western analysis showed that c-Fos and JunB bind to the AP1
element during hypoxia and that these protein levels are stimulated by
hypoxia. Mutation of the AP1 sequence prevented stimulation of
transcription of the TH-chloramphenicol acetyltransferase reporter gene
by hypoxia.
Cells in mammalian tissues are primarily aerobic and therefore
highly dependent upon a continuous supply of oxygen. A primary
mechanisms by which mammals respond to reduced O (hypoxia)
is hyperventilation, which enhances the delivery of O
to
cells by increasing arterial O
tension. The
hyperventilation that occurs during hypoxia is mediated by the
O
-sensitive (type I) cells in the carotid body. The carotid
body is located bilaterally at the bifurcation of the carotid artery
and releases dopamine in response to a reduction in arterial O
tension. The activity of tyrosine hydroxylase (EC 1.14.16.2, TH), (
)the rate-limiting enzyme in the biosynthesis of dopamine,
is enhanced in the carotid body (3) and adrenal gland (4) during hypoxia. We reported recently that hypoxia enhances
TH gene expression in the rat carotid body (1) and stimulates
both the rate of TH gene transcription and TH mRNA stability in
pheochromocytoma (PC12) cells(2) . We use the PC12 clonal cell
line as a model system to study the molecular genetic mechanisms that
regulate TH gene expression during hypoxia. Transcriptional studies
using nested deletions of the proximal promoter region of the TH gene
linked to a reporter gene revealed that the O
-responsive
sequences reside within a fragment that extends from -272 to
+ 27 bases, relative to transcription start site(2) .
In the present study experiments were undertaken to further
characterize the sequences and trans-acting protein factors
that regulate the rate of transcription of TH gene during hypoxia. We
report here that the O responsiveness of the TH gene is
mediated by a short fragment of the proximal promoter that contains the
AP1 and HIF-1 (hypoxia-induced factor binding site) sequences. Findings
from immunological studies revealed that c-Fos and JunB bind to the AP1
element during hypoxia. In addition, we report that mutation of the AP1
element abolished the transcription response to hypoxia.
We showed previously that a fragment of the TH 5`-flanking
region that extends from -272 to +27 contains the elements
necessary to regulate increased transcription during
hypoxia(2) . To further localize the hypoxia-responsive
sequences on the TH gene, PC12 cells were transiently transfected with
plasmid constructs consisting of various lengths of 5` TH flanking
sequences (-284, -150, -110, -91, or -37
to +27) linked to a CAT reporter gene, and then exposed to either
21% O (normoxia) or 5% O
(hypoxia) for 24 h.
Results from these assays, which were performed in triplicate, revealed
an oxygen-responsive region that is located between -284 and
-150 of the TH gene (Fig. 1). PC12 cells transfected with
the -284/+27 TH-CAT construct showed a marked increase in
transcriptional activity during hypoxia, whereas constructs consisting
of truncated promoter regions (-150, -110, -91, and
-37) failed to mediate an increased rate of transcription during
hypoxia.
Figure 1:
Transcription analysis of transfected
recombinant TH-CAT constructs to identify the O-sensitive
region of the TH gene. Chloramphenicol acetyltransferase assays were
performed on lysates from PC12 cells that were transfected with nested
deletions of TH proximal promoter and exposed to either 21% O
(open) or 5% O
(solid). Averaged
results from three separate experiments are presented by percent
acetylation of [
C]chloramphenicol during
normoxia and hypoxia. The data were normalized to the percent
acetylation of RSV-CAT, which was set arbitrarily at 100%. The increase
in activity during hypoxia that was observed with the -284 TH-CAT
construct was abolished with the -150 TH-CAT construct. Shorter
fragments (-110, -91, and -37) were not
O
-responsive. These findings indicate that the
hypoxia-responsive region is located between -284 and -150
of the 5`-flanking region of the gene.
The O-responsive -284/-150 fragment
of the TH promoter contains several cis-regulatory elements
(AP1, AP2, and HIF-1) that are activated by environmental stimuli to
regulate transcription(8, 10) . Gel mobility shift
assays were used to identify cis-acting elements that interact
with hypoxia-induced protein factors. A fragment of the native TH gene
that extends from -284 to -190 and contains the AP1, AP2,
and HIF-1 sites was generated by restriction digestion of the
-284/+27 TH-CAT construct. This fragment was then
radioactively labeled and used as a probe in gel shift experiments. The
probe was incubated with nuclear protein extracts from PC12 cells that
had been exposed to either 21% O
or 5% O
prior
to electrophoresis. Results from a typical gel shift are shown in Fig. 2. A marked increase in binding activity was observed with
extracts from cells exposed to 5% O
(lane3) as compared to that found with extracts from cells
exposed to 21% O
(lane2). There was no
shift in probe mobility in the absence of nuclear protein (lane1). Competition experiments using non-labeled
oligonucleotide probes that contain sequences that correspond to the TH cis elements, AP1 (lanes4 and 5)
and HIF-1 (lanes6 and 7), were performed to
determine if these motifs are involved in the hypoxia-induced protein
binding interaction with the TH gene. We found that probes that
contained the TH-AP1 and TH-HIF-1 sequence each partially blocked
binding activity to the native TH gene fragment during hypoxia.
However, it is important to note that there was still a
hypoxia-inducible increase in binding, but the binding activity in the
presence of either the AP1 or HIF-1 oligonucleotide probe was much less
than observed in the absence of these probes. It is also worth noting
that the AP1 and HIF-1 oligoprobes appeared to block different a part
of the DNA-protein complex. This finding indicates that hypoxia-induced
proteins interact with the -284/-190 fragment of the native
TH gene and that hypoxia-induced binding occurs at both the AP1 and
HIF-1 elements.
Figure 2:
Increased protein binding to the
5`-flanking region of TH gene during hypoxia. Gel shift assay using
nuclear protein extracts (NPE) from PC12 cells exposed to
either normoxia (C, 21% O) or hypoxia (H,
5% O
). The fragment of TH DNA corresponding to sequences
from -284 to -190 of the 5` region was used as probe.
DNA-protein complexes were resolved on 4% polyacrylamide gels in TBE
buffer. Hypoxia induced protein binding to this region of the TH gene (lanes2 and 3). There was not shift in
probe mobility in the absence of nuclear protein extract (lane1). Binding to the -284/-190 fragment was
competed with excess nonlabeled oligonucleotides corresponding to
either the TH AP1 (lanes4 and 5) or the TH
HIF-1 element (lanes6 and 7). Competition
of the binding complexes differed depending on the unlabeled
oligonucleotide (TH-AP1; HIF-1) added (+) to the binding reaction.
Interaction of proteins at both sites were
found.
To further characterize protein interactions with the TH-AP1 and TH-HIF-1 elements during hypoxia, mobility shift assays were performed with double-stranded oligonucleotide probes that contained sequences for either the AP1 or HIF-1 element. There were no observed increases in binding activity when protein extracts from PC12 cells exposed to hypoxia were incubated with the oligonucleotide probe that contained the TH-HIF-1 binding site (data not shown). Since binding to the native fragment was partially competed by an HIF-1 oligonucleotide, we were surprised that binding to the AP1 oligonucleotide was not induced by hypoxia. The reason for this is unclear, but could be due to a requirement for additional flanking sequences or to interactions with proteins that bind to other elements. We did, however, measure hypoxia-induced binding to the TH-AP1 oligonucleotide (Fig. 3), which was blocked by addition of unlabeled TH-AP1 oligonucleotide (arrow). The figure shows the same experiment that was repeated with nuclear protein extracts from two separate experiments. Nonspecific oligonucleotides failed to block binding to the TH-AP1 oligonucleotide (data not shown), which demonstrates the specificity of hypoxia-induced binding at this site. The lower band was blocked by nonspecific oligonucleotides, which indicates that this band is due to nonspecific binding activity.
Figure 3:
Increased binding activity to the TH-AP1
element during conditions of hypoxia. Gel shift assays using a
double-stranded oligonucleotide that corresponds to the TH-AP1 element
were performed with extracts from cells exposed to either 21 or 5%
O. Increased binding activity (arrow) was observed
with two different sets of nuclear proteins extracted from cells
exposed to hypoxia (5% O
). Unlabeled TH-AP1 oligonucleotide (50X) completely abolished activity. The lower band in the
complex was competed by unlabeled nonspecific oligonucleotides, which
indicates that this binding is due to nonspecific binding
activity.
We
next examined the effect of different durations of hypoxia on protein
binding to the AP1 element. Fig. 4shows a progressive increase
in binding activity to the AP1 element with nuclear protein extracts
from cells that were exposed to increasing durations (1-24 h) of
5% O. A graphical representation of binding activity from
optical density measurements of the DNA-protein complex at each
duration of hypoxia exposure is presented in Fig. 4B.
The openbar (time 0) represents binding activity to
the AP1 probe in 21% O
. Maximal binding activity was
observed with extracts taken from cells exposed to 5-6 h of
hypoxia (Fig. 4B). A notable finding was that increased
binding activity was still evident with extracts from cells exposed to
reduced oxygen for 24 h.
Figure 4:
Binding activity to the TH-AP1 element
increases and remains elevated during prolonged exposures to hypoxia. A, nuclear protein extracts from PC12 cells exposed to
increasing duration of hypoxia (5% O) bind to the TH-AP1
oligonucleotide. Increased binding activity was observed as early as
after 1 h of exposure to hypoxia; maximal activity occurred between 5
and 6 h of hypoxia. An important finding was that elevated binding was
observed as long as 24 h of hypoxic exposure. B, graphical
analysis of binding activity (-fold change) at the AP1 element that
occurred during prolonged exposure to hypoxia. Time 0 (openbar) represents control conditions (21%
O
).
We also performed a series of experiments (n = 3) to determine if binding activity is is
regulated by the level of O tension (Fig. 5). Cells
were exposed to either 10% O
or 5% O
for 1, 3,
or 5 h prior to extraction of nuclear proteins and performance of gel
mobility shift assays. Findings from these experiments demonstrates
that even modest levels of hypoxia (10% O
) can stimulate
binding activity and that binding activity is increased more rapidly in
response to more severe hypoxia (5% O
). These data are
presented graphically in Fig. 5B. Results from this
experiment demonstrate that hypoxia-induced binding to the AP1 element
is regulated by the severity of hypoxia and that mild hypoxia is
sufficient to stimulate protein binding to the AP1 element.
Figure 5:
Effect of oxygen concentration on nuclear
protein binding to the TH-AP1 element. A, PC12 cells were
exposed to either 10% O or 5% O
for increasing
lengths of time (h = hours) prior to extraction of
nuclear proteins and performance of gel shift assays. Increased binding
activity to the TH-AP1 element was observed with extracts from cells
exposed to both 10 and 5% O
. This demonstrates that a
modest level of hypoxia is sufficient to induce an increase in binding
activity of the TH-AP1 element and that binding activity is further
increased with more severe hypoxia. B, densitometric values
that show the relationship of O
level and binding activity
to the AP1 element.
To identify the protein factors that interact with the TH AP1 site, UV cross-linking experiments were first performed. Cross-linking experiments revealed that a protein complex interacts with the AP1 element, although there appeared to be no apparent qualitative differences between hypoxic and normoxic extracts. The molecular masses of the four proteins involved in the complex range from 20 kDa to about 85 kDa, as shown in Fig. 6, though the smaller molecular mass proteins might be due to degradation of protein extracts. A number of regulatory transcription factors that interact with the AP1 fall within the observed molecular mass values of the protein complex binding to the TH AP1 element. Several members of the Fos and Jun family of proteins are potential candidates in this complex (c-Fos, mass = 62 kDa; c-Jun and JunB, mass = 39 kDa).
Figure 6:
Identification of TH-AP1/protein complexes
using UV cross-linking. DNA-protein binding reactions were performed
with extracts from cells exposed to normoxia (21% O, C) or hypoxia (5% O
, H) and a
radioactively labeled AP1 oligonucleotide. Samples were irradiated with
UV light, boiled in SDS electrophoresis buffer, and electrophoresed in
a 10% stacking gel. An autoradiographic exposure to the radioactive
revealed a complex of proteins binding to the TH-AP1 element. The
proteins ranged in size from about 20, 40, and 60 kDa to 90 kDa.
Molecular sizes of the proteins were estimated by comparison of
migration with known protein standard markers. Broad estimates of sizes
were used only to determine possible known proteins interacting in the
complex. The lower molecular weight protein signal may be due to
degradation of protein extract.
Immunological assays
were next performed to attempt to identify proteins that are induced by
hypoxia and bind to the AP1 site. These assays employed the use of
antibodies against several members of the Fos/Jun family of
transcription factors, which are within the molecular weight range
identified by UV cross-linking and known to interact with the AP1
element. Results from an experiment in which the antibody against c-Fos
was used is shown in Fig. 7. A supershifted band was only
detectable with protein extracted from cells exposed to 5% O (Fig. 7A). This finding indicates that c-Fos
binds is induced by hypoxand and binds to the TH-AP1 sequence. We next
performed supershift assays using antibodies against c-Jun.
Surprisingly, no supershifted band was observed (Fig. 7B). This result was confirmed by shift-Western
analysis, which revealed the presence of c-Jun in the protein extract,
but not in the AP1-protein binding complex (data not shown). In
contrast to our finding with c-Jun, we found a very prominent
supershifted band when antibodies against JunB were used (Fig. 7B). The quantity of JunB in the binding complex
appeared to increase during hypoxia. The binding of JunB to the AP1
element during hypoxia was confirmed by shift-Western analysis (data
not shown). These findings clearly demonstrate that binding to the AP1
complex is increased during hypoxia and that this increased binding
involves both c-Fos and JunB.
Figure 7:
Supershift assays reveal the presence of
c-Fos and JunB in the AP1 binding complex. A, an antibody
against c-Fos that was incubated with the gel shift binding reaction
produced a supershifted band (solidarrow) that
migrated above the normal shifted complex (openarrow). This supershifted band indicated the presence of
c-Fos in the AP1 binding complex. c-Fos was undetectable in protein
complexes from cells that were grown in normoxia (21% O),
which indicates the induction of c-Fos during conditions of hypoxia (5%
O
). B, an antibody against c-Jun did not produce a
supershifted band in the gel mobility shift assay. Higher
concentrations (2-6 µg) also failed to produce a supershifted
band (data not shown). This result indicated that c-Jun was not present
in the TH-AP1 binding complex. Antibodies against JunB, on the other
hand, resulted in a supershifted band, which indicated that JunB is
present in the hypoxia-induced AP1 binding complex (panel
C).
To determine if DNA-protein
interactions at the TH-AP1 are responsible for increased transcription
of the TH gene during hypoxia, CAT assays were performed using a
-272 TH-CAT plasmid with a mutated AP1 element. This mutation
completely destroyed the AP1 recognition site(5) . Fig. 8shows results from a CAT assay performed with the intact
TH-AP1 and AP1 mutant constructs that had been transfected into PC12
cells prior to exposure to 5% O. Hypoxia stimulated
expression of the construct that contained the intact AP1 element (lane1), but not in the construct with the mutant
AP1 element (lane2). These results, taken together
with the protein binding data, demonstrate the importance of the AP1
element in regulation of the TH gene during hypoxia.
Figure 8:
Mutation of the AP1 element abolishes the
hypoxic induction of TH gene expression. CAT assays using PC12 cells
transfected with the wild-type -272 TH-CAT construct (lane1) or the AP1 mutant -272 TH-CAT construct (lane2). This experiment was performed in
triplicate. Cells transfected with the respective constructs were
exposed to 5% O, and CAT assays were performed. Lane2 demonstrates the abolished hypoxia-induced increase in
TH gene expression with the mutated AP1 site, in
comparison.
In the present study, we demonstrated that enhanced
transcription of the TH gene during hypoxia is regulated by specific
sequences located in the 5`-flanking region of the TH gene. Results
from transient transfection experiments revealed a critical region of
the TH gene that extends from -284 to -150, relative to
transcription start site, that is required for transcriptional
activation by hypoxia. This region of the TH gene contains a number of
well characterized transcriptional regulatory elements, including an
AP1, AP2, OCT/POU, and the more recently described HIF-1
element(8) . The HIF-1 element has been implicated in
transcriptional regulation of the erythropoietin gene during
hypoxia(11) . Moreover, members of the Fos and Jun
proto-oncogene family interact with the AP1 sequence and are known to
be induced by reduced O(12) . Our current findings
show that hypoxia-induced proteins bind to the TH gene fragment that
contains the HIF-1 and AP1 elements. Semenza and Wang (8) recently demonstrated the existence of a protein induced by
hypoxia (HIF-1), which binds to its own consensus element to regulate
erythropoietin gene expression during hypoxia. Others have reported
changes in binding activity to the AP1-like motif of the TH gene during
different environmental stimuli(13, 14, 15) .
We therefore focused on these two regulatory elements in our effort to
further characterize the molecular mechanisms that regulate
transcription of the TH gene during hypoxia.
Unlabeled
oligonucleotides that corresponded to the HIF-1 and AP1 elements were
used as competitors in mobility shift assays. Both the HIF-1 and AP1
oligonucleotides abolished binding to the native gene fragment that
extended from -284 to -190. These results suggest that
protein interactions with the TH gene during hypoxia occurred at the
AP1 and HIF-1 sites. Although the HIF-1 element has been implicated in
the regulation of genes during hypoxia(8) , this is the first
evidence for protein binding activity at an AP1 element as a step in
the cascade of transcriptional regulatory events during hypoxia.
Mobility shift assays were also performed with oligonucleotide probes
that contained either the AP1 and HIF-1 to determine if binding to
these individual elements is enhanced binding during hypoxia. Binding
activity was shown to be increased at the TH AP1 motif, but not at the
HIF-1 element. Since oligonucleotide probes were used in these
experiments, critical flanking sequences required for HIF-1 binding
might not have been included in the probe. Our finding that an HIF-1
competitor sequence successfully competes for binding to the native DNA
fragment certainly supports the possibility of protein binding to this
element during hypoxia. It is also possible that failure of hypoxia to
enhance binding activity to the HIF-1 oligonucleotide was due to the
relatively mild conditions of hypoxia (5% O) in our
experiments, as compared to the conditions (1% O
) used in
studies in which the HIF-1 element and protein were
characterized(8) . In contrast, binding to the AP1 element was
markedly enhanced even during mild (10% O
) and moderate (5%
O
) hypoxia. Another notable result from the current study
was the sustained binding to the AP1 oligonucleotide probe when protein
was extracted from cells that had been exposed to relatively long (24
h) periods of hypoxia. This is potentially a very important observation
for understanding the molecular mechanisms involved in sustained TH
gene expression in O
-sensitive cells during chronic
hypoxia, which is the case with most pathological and physiological
conditions associated with reduced O
tension .
Several trans-acting protein factors are known to be induced during
hypoxia, including members of the Fos and Jun families of transcription
factors (12) NFB(16) , and HIF-1(8) . It
has also been shown that NF-
B and the Fos/Jun families of proteins
are capable of binding to the AP1 consensus element to regulate gene
expression(32) . Our results show that hypoxia-inducible
binding to the AP1 element is specific, and that binding to this
element remain elevated during prolonged exposures to hypoxia (24 h).
Findings from mobility shift experiments indicate that proteins bound
to the AP1 element may play an important role in control of
transcription of the TH gene during low oxygen conditions. This is
supported by our finding that mutation of the AP1 element abolishes
transcriptional activation of the TH-CAT transgene during hypoxia.
Regulation of gene expression involving the AP1 element has been studied extensively and involves the interactions of family members of the Fos and Jun proto-oncogenes, as well as their complex interactions with other members of the leucine zipper family of DNA-binding proteins (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) . Immunological approaches were employed to help identify members of the Fos and Jun families that bind to the TH-AP1 element during hypoxia. Western blots showed that c-Fos, c-Jun, and JunB proteins are induced in PC12 cells during hypoxia (data not shown). The same antibodies were then used in supershift assays to determine which proteins were involved in the AP1 binding complex. Results from these experiments revealed that c-Fos and JunB, but not c-Jun, bind to the AP1 element during hypoxia. Our results also suggest that these proteins were more abundant extracts from cells exposed to hypoxia. Shift-Western assays confirmed the result that c-Fos and JunB bind to the AP1 element during hypoxia, with the exclusion of c-Jun. This finding suggests that a selective complex formation occurs between the members of the Fos/Jun family (c-Fos/JunB) of trans-acting protein factors that bind the TH-AP1 element during hypoxia.
Fos and Jun proteins can form
dimers with other members of the bZIP class of proteins through leucine
zipper
interactions(18, 19, 20, 21, 22, 23, 24, 25) ,
and with the transcription factor NF-B(17) . It has been
suggested that these proteins are expressed in a stimulus-specific and
cell-specific manner with the subsequent formation of specific
heterodimers(26, 27, 28, 29) . It
has long been accepted that c-Jun/AP1 is capable of binding the
specific heptamer known as TRE/AP1, and similar
variants(30, 31) . It is well documented that Jun
proteins can form heterodimers with the trans-acting factor
c-Fos(32) . However, recent research has revealed that members
of the Fos and Jun families interact in different combinations, as well
as with other leucine zipper proteins to form heterodimers that
interact with the AP1 element on target genes. For example, Diamond and
co-workers (33) reported that the function of glucocorticoid
receptor as a positive or negative regulator was determined by the
ratio of Fos and Jun proteins present. Thus it appears that the
functions of Fos and Jun are dependent on a number of factors,
including, the presence of other interacting trans factors,
cellular milieu, cell type, and the type of environmental stimuli. In
the present study, we observed specific interactions of c-Fos and JunB
at the AP1 element of the TH gene during hypoxia.
Since Fos and Jun interactions are dependent on cell type and the environmental stimulus, there may be active selection of JunB over c-Jun in the TH-AP1 binding complex in PC12 cells during hypoxia. This particular choice is interesting in that JunB has been identified as a repressor of transcriptional activation by c-Jun(27) . Chiu et al.(27) suggested that this type of ``feed-back'' regulation is extremely valuable in a system where proteins, like Fos and Jun, are co-induced by environmental stimuli. Others have shown the involvement of c-Fos and c-Jun binding at the TH-AP1 element during various stimuli, including cold stress (14) and 12-O-tetradecanoylphorbol-13-acetate treatment(15) , while nerve growth factor treatment of PC12 cells has been shown to involve the binding of FosB at the AP1 site(13) . We believe that the hypoxia-induced transcription of tyrosine hydroxylase in oxygen-sensitive cells, such as PC12 and carotid body type I cells, involves a unique AP1 transcriptional complex of c-Fos and JunB. It should be noted that the TH-AP1 element is not consensus and that this may contribute to binding of specific heterodimer pairs. These data taken together suggest yet another mechanism of transcriptional regulation in response to environmental stimuli by varying the interacting trans factors and their target cis elements.