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Vol. 56, Issue 6, 1127-1137, December 1999
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina
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Summary |
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 Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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The aryl hydrocarbon nuclear translocator (ARNT) protein functions as a transcription factor after dimerization with other basic helix-loop-helix proteins. Thus, dimerization of ARNT within one pathway may limit the availability of this protein to others. To investigate this issue, aryl hydrocarbon receptor (AHR)-mediated signaling was investigated in mouse (Hepa-1), rat (H4IIE), and human (HepG2) hepatoma cell lines undergoing physiologically induced hypoxia (<1% O2). Basal levels of ARNT protein were not affected by hypoxia in any cell line, and ARNT remained exclusively nuclear. Furthermore, quantitative Western blotting revealed that the concentration of ARNT sequestered during hypoxia represented a small fraction of the total ARNT protein pool (12 and 15% in Hepa-1 and H4 cells, respectively). When the AHR-mediated signaling pathway was activated during hypoxia by 2,3,7,8-tetrachlorodibenzo-p-dioxin, the induction of P4501A1 protein was reduced by 55% without changes in the level of mRNA in Hepa-1 cells, whereas the levels of induction of both P4501A1 protein and CYP1A1 mRNA were reduced by >80% in the H4 cell line. Importantly, gel mobility shift analysis and Western blotting showed that the same level of AHR/ARNT complexes could be detected in cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin during hypoxia and normoxia. These data suggest that the effects of hypoxia on AHR-mediated gene regulation occur distal to the formation of AHR/ARNT complexes and imply that functional interference between hypoxia and AHR-mediated signaling does not occur through competition for ARNT protein.
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Introduction |
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Top
Abstract
 Introduction
 Materials and Methods
Results and Discussion
References
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The
aryl hydrocarbon nuclear translocator [ARNT/hypoxia factor-1
(HIF-1)] protein is a member of the basic
helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) family of transcription
factors (Huang et al., 1993
; Whitlock, 1993; reviewed in Hankinson,
1995). ARNT protein is constitutively expressed in all cell culture
models evaluated to date and is localized exclusively within the
nucleus of these cells (Pollenz et al., 1994; Holmes and Pollenz,
1997). The ARNT protein does not appear to bind a ligand or require
activation but functions as a heterodimeric binding partner for other
bHLH/PAS proteins to mediate several signal transduction pathways. ARNT forms a heterodimer with the aryl hydrocarbon receptor (AHR), and the
AHR/ARNT complex associates with the xenobiotic-responsive elements
(XREs) to mediate many of the biological effects of halogenated aromatic hydrocarbons (Whitlock, 1993; Hankinson, 1995). In addition, ARNT forms a heterodimer with HIF-1
to regulate hypoxia-inducible genes such as vascular endothelial growth factor, erythropoietin, and
numerous glycolytic enzymes and transporters (reviewed in Bunn and
Poyton, 1996; Semenza, 1998). ARNT also appears to regulate genes
through the CACGTG E-box element (Antonsson et al., 1995; Sogawa et
al., 1995; Swanson et al., 1996) and can interact with mouse SIM
(Ema et al., 1996; Moffett et al., 1997). Thus, ARNT appears to be a
protein critical to the function of at least three distinct signaling
pathways that are activated by different stimuli. Indeed, the
expression of ARNT appears to be essential for normal development in
the mouse because a genetic knock-out of the ARNT gene results in the
death of animals by gestation day 10 (Kozak et al., 1997; Maltepe et
al., 1997). Therefore, it is of interest to determine 1) how
ARNT-dependent pathways function when more than one is activated, 2)
whether one pathway is dominant over the others, and 3) whether some of
the biological affects observed after exposure to compounds such as
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are related to
recruitment of ARNT to the AHR-mediated signaling pathway and away from others.
To begin to address this question, it is critical to know the
concentration and subcellular location of ARNT in cells so the amount
used by each signaling pathway can be calculated. Recent studies from
this laboratory have used quantitative Western blotting procedures to
show that the concentration of ARNT in nine different cell culture
lines averages ~156 fmol/mg cell lysate (~21,000 ARNTs/cell; Holmes
and Pollenz, 1997). In total tissue lysates derived from the spleen,
liver, and thymus of female Sprague-Dawley rats, ARNT levels were ~50
fmol/mg total tissue lysate (Pollenz et al., 1998). Thus, the ARNT
protein represents a modest 0.001 to 0.002% of total cellular protein
and appears to be susceptible to sequestration if a binding partner was
expressed at sufficiently high levels or if the level of ARNT was
reduced during signaling. Indeed, the concentration of AHR protein in
the same cell lines used to evaluate ARNT averaged >700 fmol/mg cell
lysate (Holmes and Pollenz, 1997). These findings established the
hypothesis that saturating levels of ligand might activate the entire
pool of AHR resulting in binding to the entire pool of ARNT and
inhibition of other ARNT-dependent signaling pathways. However, such a
scenario does not appear likely because the vast majority of liganded
AHR is rapidly degraded both in vitro and in vivo without affecting the
level of ARNT protein (Giannone et al., 1995; Pollenz, 1996; Pollenz et
al., 1998; Roman et al., 1998). Thus, although the AHR is 4- to 10-fold
higher than ARNT in vivo and in vitro, only ~15% of the ARNT pool is
used when the AHR-signaling pathway is saturated. Similar studies with
HIF-1 have not been performed; thus, a current area of
research in numerous laboratories concerns the interplay of AHR and
hypoxia signaling pathways through ARNT.
In the HepG2 human hepatoma cell line, results indicate that during
hypoxic signaling stimulated by CoCl2, the
expression of TCDD-inducible reporter genes and the induction of the
endogenous cytochrome P4501A1 (CYP1A1) gene were reduced by
~50% (Gradin et al., 1996). It was also noted that there was a
functional reduction in the level of AHR/ARNT heterodimer as detected
by gel shift analysis and that HIF-1 appeared to have a greater
affinity for ARNT than the AHR. Similar results were observed in the
Hep3B cell line where treatment with CoCl2
reduced agonist-induced activity of XRE-driven reporter genes and
endogenous CYP1A1 by ~45% (Chan et al., 1999). In
contrast, experiments in the mouse Hepa-1c1c7 cell line showed that
AHR-mediated CYP1A1 expression and DNA binding of AHR/ARNT
heterodimers were only minimally reduced under hypoxia (Gassmann et
al., 1997). Taken together, these three reports suggest that hypoxic
signaling may partially affect gene regulation through AHR-mediated
signaling but do not provide direct insight into the mechanism of the
response. Specifically, it is unclear 1) whether similar results are
observed under physiological hypoxia (<1% O2),
2) whether the ratio of AHR to ARNT protein is important in observing
functional interference between the two pathways, 3) whether the timing
of the hypoxic signal during activation of the AHR is critical to the
biological outcome, 4) whether the hypoxic state of the cell
contributes to reduced levels of gene induction, and 5), most
important, whether ARNT is the limiting factor in the reduced response
to AHR agonists.
To answer some of these questions, studies were initiated to specifically evaluate the AHR-mediated signaling pathway under various hypoxic stimuli in cell lines that express defined concentrations of AHR and ARNT protein. AHR-mediated signaling was evaluated by measuring the induction of the endogenous CYP1A1 mRNA and P4501A1 protein, the presence of AHR/ARNT heterodimer, and the cellular concentration of AHR and ARNT protein, with a focus on whether ARNT was a limiting factor in AHR signaling during hypoxia. The results indicate that reductions in AHR-mediated gene regulation under hypoxia are not due to functional interference of hypoxia and AHR-mediated signaling through competition for the ARNT protein.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Chemicals. TCDD (98% stated chemical purity) was obtained from Radian Corp. (Austin, TX). Desferrioxamine (DFO) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals used in this study were of the highest grade available from commercial sources.
Antibodies.
Specific antibodies against the mouse ARNT (R-1)
and AHR (A-1A) have been described previously (Holmes and Pollenz,
1997; Pollenz et al., 1994; Pollenz et al., 1998). All antibodies are affinity-purified IgG fractions. Antibodies specific to -actin were
purchased from Sigma Chemical Co. Anti-HIF-1 antibodies were
generously provide by Dr. C. Bradfield (University of Wisconsin) and
Dr. L. Poellinger (Karolinska Institute). Antibodies specific for rat
P4501A1 were purchased from Zenotech (St. Louis, MO). Goat anti-rabbit
IgG conjugated to horseradish peroxidase (GAR-HRP) or Texas Red was
purchased from Jackson ImmunoResearch (West Grove, PA).
Buffers. PBS consists of 0.8% NaCl, 0.02% KCl, 0.14% Na2HPO4, and 0.02% KH2PO4, pH 7.4. The 2× gel sample buffer consisted of 125 mM Tris, pH 6.8, 4% SDS, 25% glycerol, 4 mM EDTA, 20 mM dithiothreitol, and 0.005% bromphenol blue. TBS consists of 50 mM Tris and 150 mM NaCl, pH 7.5. TTBS consists of 50 mM Tris, 0.2% Tween 20, and 150 mM NaCl, pH 7.5. TTBS+ consists of 50 mM Tris, 0.5% Tween 20, and 300 mM NaCl, pH 7.5. BLOTTO is 5% dry milk in TTBS. The 2× lysis buffer consisted of 50 mM HEPES, pH 7.4, 40 mM sodium molybdate, 10 mM EGTA, 6 mM MgCl2, and 20% glycerol.
Cell Culture Lines and Growth Conditions. Wild-type Hepa-1c1c7 cells (Hepa-1) were a generous gift from Dr. James Whitlock Jr. (Department of Pharmacology, Stanford University). These cells were propagated in Dulbecco's modified Eagle's medium supplemented with 5% FBS. All other cells were obtained from American Type Culture Collection (Rockville, MD). H4IIE cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 5% FBS. All experiments on each cell line were completed within a 2-month period.
Generation of Hypoxic Conditions. Hypoxia was generated by incubating cells in a hypoxia chamber (Billups Rothberg, San Diego, CA) perfused with a gas mixture of 1% O2/5% CO2/94% N2 for 30 min at 3 liters/min. The chamber was then sealed and placed in a CO2 incubator at 37°C for the indicated times. Hypoxia was monitored with a gas meter and was <5% O2 within 4 min and <2% O2 within 6 min of start of the gas flow. When cells were dosed with TCDD after hypoxia, the chamber was opened, TCDD was applied to the cultures, and the chamber was sealed and perfused with 1% O2 as described earlier and returned to the 37°C incubator. In these instances, cells were dosed with TCDD and returned to the chamber within 1 min.
Preparation of Total Cell and Nuclear Lysates.
Total cell
lysates for Western blot analysis were prepared by sonicating cell
pellets in 1× lysis buffer supplemented with Nonidet P-40 (0.5%)
essentially as detailed previously (Pollenz et al., 1994; Pollenz,
1996; Holmes and Pollenz, 1997). Total nuclear lysates were prepared by
vortexing cells in 1× lysis buffer supplemented with Nonidet P-40
(1%). The solution was then centrifuged for 2 min at 700g
to pellet nuclei. The supernatant (cytosol) was removed and
supplemented with an equal volume of 2× gel sample buffer, and the
nuclei were washed with 500 µl of 1× lysis buffer. Nuclei were
pelleted through centrifugation for 2 min at 700g and then
sonicated in the presence of 1× lysis buffer supplemented with Nonidet
P-40 (1%). Nuclear lysates were then combined with an equal volume of
2× gel sample buffer and heated at 95°C for 5 min. Protein
concentrations of all samples were determined with the Coomassie Plus
Protein assay (Pierce, Rockford, IL). All samples were stored at
20°C.
Gel Electrophoresis and Western Blotting. Total cell or nuclear lysates were resolved by denaturing electrophoresis on discontinuous polyacrylamide slab gels [SDS-polyacrylamide gel electrophoresis (PAGE)] and were electrophoretically transferred to nitrocellulose. Immunochemical staining was carried out with varying concentrations of primary antibody (see text and figure legends) in BLOTTO buffer supplemented with DL-histidine (20 mM) for 1 to 2 h at 22°C. Blots were washed with three changes of TTBS+ for a total of 45 min. The blot was then incubated in BLOTTO buffer containing a 1:10,000 dilution of GAR-HRP for 1 h at 22°C and washed in three changes of TTBS+ as earlier. Before detection, the blots were washed in TBS for 5 min. Bands were visualized with the enhanced chemiluminescence (ECL) kit as specified by the manufacturer (ECL Kit; Amersham, Arlington Heights, IL). Multiple exposures (autoradiographs) of each blot were produced to ensure linearity.
Quantification of AHR, ARNT, and P4501A1.
The linearity of
the R-1, A-1A, P4501A1, and -actin antibodies for detection of
target proteins has been described in detail previously (Pollenz, 1996;
Holmes and Pollenz, 1997; Pollenz et al., 1998; Roman et al., 1998). To
calculate the actual concentration of AHR or ARNT in a given sample,
known amounts of protein (i.e., nuclear lysate, total cell lysate, and
so on) were resolved by SDS-PAGE with known concentrations of Hepa-1
cell lysates that contain 2200 fmol of AHR/mg total lysate and 231 fmol
of ARNT/mg total cell lysate. Western blots were developed by the ECL
technique as described by the manufacturer (Amersham), and multiple
exposures of autoradiographs were evaluated by regression analysis as
previously detailed (Pollenz, 1996; Holmes and Pollenz, 1997; Pollenz
et al., 1998; Roman et al., 1998). Because it has previously been determined that the total amount of protein in the nuclear lysate fraction represents 25% of the total cellular protein pool (Pollenz et
al., 1994), it is possible to determine the actual amount of target
protein retained within each cellular fraction.
Electrophoretic Mobility Shift Assay (EMSA).
Oligonucleotides XRE-1 (5'-CGGCTCGGAGTTGCGTGAGAAGAG) and XRE-2
(5'-CGGCTCTTCTCACGCAACTCCGAG) were annealed and labeled with 32P-dCTP by Klenow fill-in (Sambrook et al.,
1989). The double-stranded fragment corresponds to the consensus XRE-1
of the CYP1A1 promoter as previously described (Shen and
Whitlock, 1992). From 5 to 15 µg of nuclear extract was incubated at
22°C for 15 min in 1× gel shift buffer supplemented with 80 mM KCl
and 0.1 mg/ml poly(dI/dC). In some instances, 0.5 to 1.0 µg of
affinity-purified IgG was included in the sample. Approximately 4 ng of
32P-labeled XRE was then added to each sample,
and the incubation was continued for an additional 15 min at 22°C.
The samples were resolved on 5% acrylamide-0.5% Tris/boric acid/EDTA
gels, dried, and exposed to film.
RNA Isolation and Northern Blot Analysis.
Total RNA was
isolated from cells with the RNAeasy kit essentially as detailed by the
manufacturer (Qiagen, Studio City, CA). RNA was resolved on
formaldehyde gels and transferred to nitrocellulose via vacuum
blotting. Blots were probed with 32P-labeled cDNA
fragments, washed, and exposed to film as described previously
(Sambrook et al., 1989).
Cell Growth Assay.
Stock cells were harvested, and
105 cells were then plated onto 12 dishes of 60 mm and allowed to attach for 12 h. After this time period, cells
were harvested from four plates, counted, and lysed with 1 M NaOH to
assess the total cellular protein. Four plates were then placed under
hypoxia (1% O2) or normoxia for 19 to 24 h.
After the incubation, cells were counted and lysed as above. Cell
growth rates (doubling time) was then determined by as Doubling time
(h) = 0.693/[ln cell count at harvest ln cell count at
time 0/total growth time (h).
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Results and Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Hypoxia Stimulation Does Not Affect Location or Concentration of
ARNT Protein in Hepa-1 Cells.
Studies in vitro have demonstrated
that HIF-1 protein appears to have a higher affinity for ARNT than
the AHR (Gradin et al., 1996); thus, it has been hypothesized that
there may be competition for the ARNT protein when hypoxia and
AHR-mediated signaling pathways are simultaneously stimulated (Gradin
et al., 1996; Chan et al., 1999). However, the validity of this
hypothesis requires an understanding of 1) how hypoxia affects the
basal level of ARNT protein in cells, 2) the importance of timing in
the stimulation of both pathways, and 3) calculation of the actual
amount of ARNT protein used by both signaling pathways. To answer these
questions, it was first necessary to establish that the level of ARNT
present in nuclear lysates was an accurate measure of hypoxic signaling
and correlated to the presence of HIF-1 .
protein present in nuclear lysates and cytosol were then
determined by Western blotting. A representative experiment is shown in
Fig. 1. The blots show that HIF-1
protein is predominately present in the nuclear lysate fraction of
hypoxic cells and is not detected at high levels in the cytosolic
fraction (Fig. 1A). The ARNT protein is also detected in the nuclear
fraction of hypoxic cells but is detected in the cytosolic fraction as
well (Fig. 1B). Importantly, the level of both ARNT and HIF-1
protein detected in the nuclear lysate fraction is markedly decreased
as hypoxic cells are returned to normoxia. However, although
HIF-1 is no longer detected in any fraction, ARNT protein is present
in the cytosol at the same level as in normoxic controls (Fig. 1B). It
is important to note that previous immunological studies have
demonstrated that ARNT is localized exclusively within the nucleus of
Hepa-1 cells and that only ARNT tightly associated with nuclear
structures (such as AHR/ARNT complexes associated with XREs) is
retained in the nucleus after lysis (Pollenz et al., 1994; Pollenz,
1996; Holmes and Pollenz, 1997). Indeed, immunological staining of
fixed cells shows that the subcellular location of ARNT is exclusively
nuclear before, during, and after hypoxic treatment (Fig. 1C). Thus,
these results are consistent with previous findings suggesting that the
basal level of ARNT protein is unaffected by hypoxia (Pugh et al.,
1997; Salceda and Carol, 1997; Huang et al., 1998). To confirm this
hypothesis, total cell lysates were prepared from Hepa-1 cells that
were incubated under hypoxia (1% O2) for 16 h or incubated under hypoxia for 16 h and returned to normoxia for
an additional 30 min. Equal amounts of protein were evaluated for
ARNT and -actin by Western blotting. Figure 1D shows that the level
of ARNT protein remains constant during and after hypoxic stimulation.
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and an active hypoxic
signaling pathway (the hypoxia-responsive genes aldolase and glucose
transporter 1 were also induced 4- to 6-fold in hypoxia-stimulated
Hepa-1 cells). Third, the subcellular location of ARNT is unaffected by
hypoxia, and therefore interaction with HIF-1 must occur in this
compartment. Last, the rapid loss of HIF-1, but not ARNT, from the
nuclear lysate fraction under normoxia indicates that HIF-1 and ARNT
protein levels are regulated distinctly in the Hepa-1 cell line. Thus,
ARNT present in the nuclear lysate fraction likely represents
ARNT/HIF-1 complexes, whereas cytosolic ARNT represents that
fraction of the ARNT protein pool that has redistributed from the
nucleus and is an approximation of ARNT protein pool that may be
available to other dimerization partners (if the entire pool is
accessible). The rapid loss of HIF-1 after normoxia is consistent
with the hypothesis that HIF-1 protein concentration is controlled
primarily at the level of protein stability and is rapidly degraded
under normoxic conditions (Pugh et al., 1997; Salceda and Carol, 1997;
Huang et al., 1998). Interestingly, these findings parallel the
interaction of the AHR and ARNT during AHR-mediated signaling, where
AHR is rapidly degraded after ligand binding without changes in
concentration or subcellular localization of ARNT (Pollenz, 1996;
Pollenz et al., 1998; Roman et al., 1998).
Small Fraction of ARNT Protein Pool Is Used during Hypoxic
Signaling in Hepa-1 and H4IIE Cells.
Having established that ARNT
detected in nuclear lysate fractions of hypoxic cells correlated to the
presence of HIF-1, studies were next focused on the level of the
ARNT pool used during physiologically induced hypoxia and the time
course of the response. For these studies, mouse Hepa-1 and rat H4IIE
(H4) cells were used. The choice of the H4 cell line was based on a
number of criteria. First, the concentration of AHR in H4 cells (70 fmol of AHR/mg total cell lysate; Holmes and Pollenz, 1997) is closer
to the concentration of AHR in rat liver (236 fmol of AHR/mg liver
lysate; Pollenz et al., 1998) than the Hepa-1 cell (2300 fmol of AHR/mg cell lysate; Holmes and Pollenz, 1997) and may represent a more physiologically relevant model system for analysis of AHR-mediated signaling. Second, the molecular mass and biochemical properties of the
AHR expressed in H4 cells are more consistent with other mammalian AHRs
than the AHR expressed in Hepa-1 cells (Poland and Glover, 1990;
Dolwick et al., 1993). Finally, the H4 cell represents a hepatic model
in which the AHR/ARNT ratio is <1.0 as opposed to 10 in the Hepa-1
cell line (Holmes and Pollenz, 1997). Thus, it becomes possible to
evaluate the importance of AHR and ARNT protein concentration on
AHR-mediated signaling during hypoxic stimulation.
-actin by Western blotting.
Figure 2 shows a representative blot from each cell line. Hepa-1 cells incubated in 1% O2
show significant levels of ARNT protein in nuclear lysates within
2 h of treatment that are maintained at a similar level for the
duration of the hypoxic stimulation (Fig. 2A). In the H4 cell line,
significant levels of ARNT are also detected within 2 h of hypoxia
and are maintained for the duration of the stimulation (Fig. 2B). In
addition, as observed for the Hepa-1 cell line (Fig. 1B), the level of
ARNT protein in nuclear lysates is rapidly lost from the fraction when cells are returned to normoxia. Thus, it appears that these cells sequester a fraction of the ARNT protein pool during hypoxia and maintain the level used as long as hypoxia is maintained. Similar results were obtained when mouse 10T1/2 fibroblasts and human HepG2
hepatoma cells were evaluated in identical experiments (R. S. Pollenz, unpublished results).
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; Holmes and Pollenz, 1997; Roman et al., 1998). Previous studies
have established that a Hepa-1 cell contains ~33,000 molecules of
ARNT, whereas H4 cells contain ~19,000 molecules (Pollenz, 1996;
Holmes and Pollenz, 1997). It has also been established that the amount
of protein present in the nuclear lysate fraction of these cells
represents ~25% of total cellular protein (Pollenz et al., 1994;
Pollenz, 1996). [Nuclear lysates, cytosol, and total cell lysates were produced from a defined number of cells. Protein assays revealed that
the percentage of cellular protein contained within the nuclear lysate
fraction was ~25% of the total and that the remaining protein could
be recovered in the supernatant (cytosol) fraction (Pollenz et al.,
1994).] Therefore, it is possible to determine the concentration of
ARNT protein used during hypoxia by comparing the amount of ARNT in a
known concentration of nuclear lysate with that of the total cell. The
results indicate that the amount of ARNT sequestered in Hepa-1 cells
after the exposure to physiological hypoxia represents 12 ± 5% of the total ARNT protein pool (~4000 molecules). For H4 cells,
the data show that the amount of ARNT sequestered after hypoxia
exposure represents 15 ± 5% of the total ARNT pool (2800 ARNT
molecules). Thus, the results of these experiments indicate that the
concentration of ARNT tightly associated with nuclear structures (i.e.,
associated with HIF-1 or other isoforms) under physiological hypoxia
is a relatively small fraction of the total ARNT pool (as shown in Fig.
1B). However, a caveat to these results is the fact that it is not
known whether the entire pool of ARNT is "accessible" to the
various dimerization partners. The calculation that ~15% of the ARNT
pool is sequestered by the AHR or HIF-1 may in fact be higher if the
entire pool is not available for dimerization.
Hypoxia Stimulation of Hepa-1 Cells Results in Reduced Levels of
TCDD-Induced P4501A1 Protein but Not CYP1A1 mRNA.
Because hypoxia-mediated signaling appeared to use modest levels of
ARNT protein pool, the next set of studies focused on whether hypoxia
affected AHR-mediated signaling through ARNT. Hepa-1 cells were
preincubated under physiological hypoxia (1% O2)
for either 2 or 16 h and then exposed to a single dose of TCDD (1 nM) for either 6 or 16 h while still under the hypoxic stimulation. Total cell lysates were prepared and evaluated for P4501A1
and -actin protein by Western blotting. Representative blots are
shown in Fig. 3. The results show that
P4501A1 levels are reduced by 55% when cells are preincubated under
1% O2 for 16 h (Fig. 3A). However,
TCDD-inducible P4501A1 protein is not reduced when TCDD treatment
occurs immediately after return of the cells to normoxia. Similar
results were observed when the hypoxic preincubation was reduced to
2 h. Thus, hypoxia resulted in reduced levels of TCDD-induced
P4501A1 protein, and the effect correlated temporally to the
recruitment of ARNT by HIF-1 (Fig. 2).
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;
Hankinson, 1995). Therefore, studies were carried out to evaluate
whether hypoxia affected the TCDD-mediated induction of
CYP1A1 mRNA. Hepa-1 cells were incubated in an atmosphere of
1% O2 for 16 h and then treated with TCDD
for 6 h while still under hypoxia. In addition, plates of cells
were removed from hypoxia after 16 h and treated with TCDD for
6 h under normoxia. Total RNA was prepared, and the expression of
CYP1A1 and -actin was determined by Northern blotting.
The results show that the induction of CYP1A1 is identical
whether cells are treated with TCDD under hypoxic or normoxic
conditions (Fig. 3B). Similar results were observed when the hypoxic
preincubation was reduced to 2 h (R. S. Pollenz, unpublished
results). These results are in sharp contrast to those observed for
induction of P4501A1 protein (Fig. 3A). It appears that the reduced
level of TCDD-inducible P4501A1 protein in hypoxia-treated cells does not correlate to the level of CYP1A1 mRNA and the
recruitment of ARNT by HIF-1. Reductions in P4501A1 appear to be
distal to the induction of CYP1A1.
Hypoxia Stimulation Does Not Affect Formation of AHR/ARNT
Heterodimers in Hepa-1 Cells.
The finding that TCDD-inducible
P4501A1 protein, but not CYP1A1 mRNA, was reduced by hypoxia
in Hepa-1 cells suggested that the formation of the AHR/ARNT
heterodimer and binding at the CYP1A1 promoter were
occurring normally in cells undergoing hypoxic signaling. To confirm
this hypothesis, Hepa-1 cells were incubated under normoxia or hypoxia,
and nuclear extracts were evaluated by EMSAs. Figure
4A shows a representative experiment. A
specific shift of similar intensity is observed in all samples treated
with TCDD regardless of whether the nuclear extracts were isolated from cells exposed to hypoxia (Fig. 4A, compare lane 3 with lane 7 and lane
4 with lane 8). Quantification of the shifted bands showed them to be
within 10% of each other when normalized to the amount of nuclear
extract used. To confirm the presence of the AHR and ARNT in the
shifted bands, IgG specific for AHR or ARNT was included in the
reaction before the addition of XRE. The specifically shifted band is
lost with the addition of specific antibodies but not with preimmune
rabbit IgG (Fig. 4A, lanes 9-11). In addition, Western blot analysis
of the identical nuclear lysate fractions used for the EMSA showed the
presence of similar levels of AHR protein in all samples treated with
TCDD (R. S. Pollenz, unpublished results).
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protein (Pugh et al., 1997; Salceda and Carol, 1997; Huang et al., 1998), it was pertinent to confirm that the technical aspects of
the experiment did not influence the EMSA results. Hepa-1 cells were
treated with DFO to induce hypoxia-mediated signaling. DFO has been
used in numerous laboratories as a chemical method of inducing hypoxia
(reviewed in Bunn and Poyton, 1996; Semenza, 1998), and studies in
Hepa-1 and H4 cells have confirmed that treatment with DFO results in
recruitment of ARNT to nuclear lysate fractions and the induction of
hypoxia-responsive genes (R. S. Pollenz, unpublished results).
Hepa-1 cells were treated with 100 µM DFO for 16 h and then
treated with TCDD (1 nM) for 0, 30, 45, 90, and 120 min. The results of
a representative EMSA are shown in Fig. 4B. A specific shift of similar
intensity is observed at each time point along the same time course. In
addition, DFO alone does not produce a gel shift. The ability to
observe specific shifts in this study supports the use of physiological
hypoxia as a stimulus (Fig. 4A) and indicates that the technical
aspects of the use of physiological hypoxia do not influence the
results. In summary, the results of these experiments are consistent
with previous studies in the Hepa-1 cell line (Gassmann et al., 1997) and support the hypothesis that the formation of AHR/ARNT complexes capable of binding DNA is not affected by conditions in which up to
15% of the ARNT protein pool has been recruited to the hypoxia signaling pathway.
Hypoxia Stimulation of H4 Cells Results in Reduced Levels of
TCDD-Induced P4501A1 Protein and CYP1A1 mRNA.
It was
next pertinent to determine whether hypoxia affected AHR-mediated
signaling in H4 cells because these cells express 30-fold lower levels
of AHR than Hepa-1 cells. H4 cells were incubated under hypoxia (1%
O2) and exposed to TCDD, and total cell lysates were evaluated for P4501A1 protein by Western blotting. The results show that TCDD-inducible P4501A1 protein is reduced by >80% when H4
cells are preincubated under hypoxia for 16 h (Fig.
5A). However, TCDD-inducible P4501A1
protein was not reduced if TCDD exposure was initiated immediately on
return of the cells to normoxia. In addition, as
observed in the Hepa-1 cells, the results did not change when the
hypoxic stimulation was reduced to 2 or 6 h before the addition of
TCDD (R. S. Pollenz, unpublished results).
|
-actin was
determined by Northern blotting. In sharp contrast to the results
observed in the Hepa-1 cell line, TCDD-inducible CYP1A1 mRNA
was reduced by ~75% whether cells were incubated under hypoxia for 2 or 16 h before TCDD treatment (Fig. 5, B and C). However,
TCDD-inducible CYP1A1 was not reduced if TCDD treatment
occurred immediately after return of the cells to normoxia (Fig. 5, B
and C). Thus, reductions in P4501A1 protein are supported by reduced
levels of CYP1A1 and suggest that hypoxia is affecting
function at the CYP1A1 promoter region in the H4 cell line.
Hypoxic Stimulation of H4 Cells Results in Reduced Levels of AHR
Protein, but Formation of AHR/ARNT Heterodimers Is Not Affected.
Because the TCDD-mediated induction of CYP1A1 mRNA was
dramatically reduced during hypoxia in H4 cells, studies were initiated to determine whether formation of the AHR/ARNT heterodimer was also
affected. To begin the analysis of this question, it was first of
interest to evaluate whether hypoxia affected the endogenous level of
AHR or ARNT protein in the H4 cells because this could account for
reductions in response to TCDD. Hepa-1 cells were incubated under
normoxia, incubated under hypoxia (1% O2) for 16 h, or incubated under hypoxia for 16 h and then returned
to normoxia for an additional 30 min. Total cell lysates were then evaluated for ARNT, AHR, or -actin. The results show that hypoxia does not significantly change the level of ARNT protein but results in
a 2-fold reduction in the level of AHR protein (Fig.
6A). This is the first evidence that
hypoxia may affect the basal level of a transcription factor and is
consistent with the response of cells to TCDD under hypoxia (Fig. 5).
|
-actin protein
by Western blotting. Figure 6C shows a representative blot.
Importantly, AHR protein is detected in all samples treated with TCDD
regardless of whether they were exposed to DFO. Because AHR protein is
not detected in cells incubated with DFO alone, the presence of AHR
protein cannot be due to the presence of the chemical or to
contamination of the nuclear extract with nonlysed cells. The
reduced level of AHR observed in samples treated with TCDD for 60 min
may represent a delay in the timing of the AHR-signaling cascade;
however, identical levels of AHR are observed in cells treated with DFO
or not treated with DFO after 80 and 120 min. The presence or ARNT in
each sample was confirmed by Western blotting, and both ARNT and AHR
were detected in the same fraction (elution at ~200 kDa), when
evaluated by gel filtration chromatography (R. S. Pollenz,
unpublished results). Collectively, this set of experiments support the
hypothesis that AHR/ARNT complexes capable of interacting with DNA are
present in H4 cells during hypoxic signaling with DFO. Similar findings were observed when hypoxia was used as the stimulus (R. S. Pollenz, unpublished results). It is important to note, however, that
given the reduction observed in TCDD-inducible CYP1A1 and
P4501A1, the functionality and timing of the formation of the AHR/ARNT
heterodimer may be dramatically affected by the hypoxic stress to the
cell. Indeed, analysis of H4 cells after 24 h of hypoxia revealed
that the doubling time of the culture increased from 11.9 ± 2 to
28.4 ± 5 h, whereas the concentration of total cellular
protein in each cell was unchanged. Thus, the H4 cell line appears
particularly sensitive to hypoxia, and this condition results in
greatly reduced transcription and translation of CYP1A1
after exposure to TCDD.
Hypoxia Does Not Affect Formation of AHR/ARNT Complexes in Human
Cell Lines.
Although the Hepa-1 and H4 cell line has been used
extensively for analysis of AHR-mediated signaling, other studies on
the interplay of AHR and hypoxia signaling have used human cell lines (Gradin et al., 1996; Chan et al., 1999). Therefore, studies were performed to assess the formation of AHR/ARNT complexes in two human
cell lines under physiological hypoxia. HepG2 hepatoma and MCF-7 breast
cancer cells were exposed to hypoxia (1% O2) or
normoxia and treated with TCDD, and nuclear extracts were evaluated by EMSA. The results of representative experiments are shown in Fig. 7. For the HepG2 cell line, a specific
shift is observed in all samples treated with TCDD regardless of
whether the nuclear extracts were isolated from cells exposed to
hypoxia (Fig. 7A, lanes 2 and 4). In three independent experiments, the
intensity of the specific shifts was 80 ± 12 relative
densitometry units for TCDD alone compared with 85 ± 15 relative
densitometry units for TCDD exposed under hypoxic conditions. To
confirm the presence of the AHR and ARNT in the shifted bands, IgG
specific for AHR or ARNT was included in the reaction before the
addition of XRE. The major shifted band was lost with the addition of
specific antibodies but not with preimmune rabbit IgG (Fig. 7A, lanes
5-7). These results are identical to those presented for Hepa-1 cells
(Fig. 4A). For the MCF-7 cell line, specifically shifted bands were also observed in all samples from TCDD-treated cells (Fig. 7B). The
relative intensity of the bands in hypoxia-treated cells (52 ± 10 relative densitometry units) were slightly reduced compared with
TCDD-treated controls (65 ± 15 relative densitometry units) in
two separate experiments, but the values were not statistically different. For both cell lines, the presence of AHR in nuclear extracts
was confirmed by Western blotting and showed no significant changes
whether isolated from cells exposed to hypoxia or normoxia (R. S. Pollenz, unpublished results). These results are consistent with the
findings in Hepa-1 and H4 cells and support the hypothesis that the
formation of AHR/ARNT complexes is not affected by the use of ARNT
during hypoxia-mediated signaling.
|
Conclusions and Implications.
The ARNT protein serves as a
dimerization partner for AHR, HIF-1, HIF-2, mouse SIM, and other
proteins in the bHLH/PAS family (Ema et al., 1996; Swanson et al.,
1996; Moffett et al., 1997; Probst et al., 1997; Pugh et al., 1997;
Semenza, 1998). The importance of ARNT-dependent signaling is
highlighted by several gene targeting studies.
ARNT
/ and
HIF/ mice exhibit an embryonic
lethal phenotype most probably caused by the lack of vascularization
mediated through HIF-1/ARNT complexes (Kozak et al., 1997; Maltepe
et al., 1997; Ryan et al., 1998). Gene targeting knock-outs of the AHR
on the other hand result in viable animals that exhibit significant
defects in immune system development, liver fibrosis
(Fernandez-Salguero et al., 1995), and mammary gland development
(Hushka et al., 1998). In addition, reduced expression of AHR in cell
culture models also suggests important endogenous functions for the AHR
signaling pathway in cell growth (Ma and Whitlock, 1996). Thus, an
important issue with regard to bHLH/PAS protein signaling is whether
recruitment of ARNT to one pathway limits the availability of this
protein for other bHLH/PAS factors. Analysis of this issue has the
potential to provide insight into the interaction of signaling pathways and the mechanism of toxicity for aryl hydrocarbons.
; Chan et al., 1999). Thus, it has been
hypothesized that these findings indicate that a limiting cellular
factor is shared by these pathways, with the ARNT protein as the most
likely candidate (Chan et al., 1999). The data presented in this report
strongly suggest that functional interference between hypoxia and
AHR-mediated signaling does not occur through competition for ARNT
protein. This hypothesis is supported by several experiments. First,
hypoxia does not affect the distribution or concentration of ARNT
protein present in either rat or mouse hepatoma cells, and the ARNT
protein that is sequestered is returned to the ARNT pool once the
hypoxic signal is removed. Second, the formation of AHR/ARNT complexes
should be the most sensitive endpoint if functional interference is
occurring through the use of ARNT (especially because the liganded AHR
becomes limiting due to rapid proteolysis). Physiological hypoxia does
not affect the formation of AHR/ARNT complexes capable of binding DNA
in four distinct cell lines from three different species even though
each cell line expresses different levels of AHR and ARNT protein.
Finally, quantitative Western blotting shows that the amount of ARNT
protein actually sequestered during physiological hypoxia represents
only 12 to 15% of the ARNT pool in Hepa-1 and H4 cells, and thus the
majority of the pool appears to be available to other protein factors.
It is important to note that even if this percentage is higher due to
the possibility that the entire ARNT pool is not available for
dimerization, the ability to detect AHR/ARNT complexes during hypoxia
indicates that AHR and HIF-1 are not competing for the ARNT protein
during simultaneous signaling and that ARNT is clearly not the limiting factor in the observed reductions in AHR-mediated signaling. How, then,
does hypoxia stimulation result in reductions in TCDD-mediated induction of CYP1A1 protein and mRNA in certain cells?
A possible answer to this question is that the formation of AHR/ARNT
complexes and binding to DNA do not guarantee that AHR-mediated signaling will proceed through gene regulation. As elegantly shown in
the steroid hormone receptor signaling cascades, coactivators may be
required to obtain maximal transcriptional response (Horwitz et al.,
1996), and such a factor may be limiting within the context of AHR or
hypoxia-mediated signaling. Alternatively, because hypoxia is a stress
response and the AHR/ARNT complexes appear to have formed normally, the
reductions in transcription and translation of the CYP1A1
gene may be related to metabolic state of the cell and a shift toward
maintenance of regulation of essential genes. Recent studies support
this view by clearly demonstrating that hypoxia can induce apoptosis
and affect cellular growth (Bunn and Poyton, 1996; Carmeliet et al.,
1998). Indeed, the H4 cell line exhibited dramatic effects of hypoxia
on CYP1A1 induction, and these results appear to correlate
with greatly reduced growth rates of this cell during hypoxic stress.
In contrast, Hepa-1 cells did not exhibit sensitivity to hypoxia either
in cellular growth or the magnitude of changes in TCDD-mediated signaling.
In summary, the results presented in this report are consistent with
previous studies that have shown hypoxia-induced reductions in
AHR-mediated gene regulation (Gradin et al., 1996; Chan et al., 1999)
but support a model in which ARNT is not the limiting factor. It
appears that the competition for ARNT is minimized in part because the
AHR and HIF-1 proteins capable of binding ARNT are kept in check by
proteolysis. In the case of AHR, ligand binding results in rapid
degradation of the majority of the protein pool (>85%), creating a
situation in which the AHR actually becomes the limiting dimerization
partner for ARNT in most cell lines (Pollenz, 1996; Pollenz et al.,
1998; Roman et al., 1998). For HIF-1, the protein is rapidly turned
over during normoxia (Pugh et al., 1997; Salceda and Carol, 1997; Huang
et al., 1998) and appears to be the limiting binding partner for ARNT
during the physiological hypoxia stimulation based on the levels of
ARNT actually used during signaling (<15% of the ARNT pool). Once
normoxia is reestablished, HIF-1 is rapidly degraded, allowing the
release of ARNT back to the pool (Fig. 1). A proteolytic mechanism for the regulation of transcription factors appears to be a developing biological theme and is important in the function of p53 (Ciechanover et al., 1994), c-JUN (Trier et al., 1994), nuclear factor-
B
(Palombella et al., 1994; Traencker et al., 1994), and glucocorticoid
receptors (Hoeck et al., 1989). The next challenge is to determine
whether biological conditions exist (i.e., developmental stages,
expression of additional ARNT partners) to perturb this model, which
appears to allow multiple bHLH/PAS signaling pathways to operate simultaneously.
| |
Acknowledgments |
|---|
We thank Dr. Lorenz Poellinger (Karolinska Institute) and Dr.
Chris Bradfield (University of Wisconsin) for the generous gift of
anti-HIF-1 antibodies. Dr. Peter Ratcliffe is acknowledged for
helpful discussions concerning the generation of hypoxic conditions for
these studies. We also thank the reviewers of the manuscript for their
helpful comments.
| |
Footnotes |
|---|
Received April 15, 1999; Accepted August 25, 1999
This work was supported in part by Grant ES08980 from the National Institute of Environmental Health Sciences. Portions of the work were presented at the Mechanisms of Toxicity Gordon Research Conference, July 1998, and the Society of Toxicology Meeting, March 1999.
Send reprint requests to: Dr. Richard S. Pollenz, Department of Biochemistry and Molecular Biology, MUSC, 171 Ashley Ave., Charleston, SC 29403. E-mail: pollenzr{at}musc.edu
| |
Abbreviations |
|---|
ARNT, aryl hydrocarbon nuclear translocator;
AHR, aryl hydrocarbon receptor;
bHLH, basic helix-loop-helix;
HIF-1, hypoxia-inducible factor-1;
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin;
PER, periodicity;
SIM, single minded;
PAS, PER-ARNT-SIM;
GAR-HRP, goat anti-rabbit
horseradish peroxidase;
ECL, enhanced chemiluminescence;
DFO, desferrioxamine;
TTBS, Tris-buffered saline with Tween 20;
CYP or P450, cytochrome P-450;
PAGE, polyacrylamide gel electrophoresis;
EMSA, electrophoretic mobility shift assay;
XRE, xenobiotic-responsive
element.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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