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Expression During Hypoxia1
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to ambient hypoxia. Initial studies
using microarray analysis of intestinal epithelial mRNA revealed that
hypoxia rapidly down-regulates PPAR- mRNA and protein in epithelial
cells in vitro and in vivo. Subsequent studies revealed that the
PPAR- gene bears a DNA consensus motif for the transcription
factor hypoxia-inducible factor 1 (HIF-1). EMSA analysis revealed that
ambient hypoxia induces HIF-1 binding to the HIF-1 consensus domain
of PPAR- in parallel to HIF-1 nuclear accumulation, and antisense
depletion of HIF-1 resulted in a loss of PPAR- down-regulation.
The PPAR- ligand pirinixic acid (WY14643) functionally
promoted IFN-
-induced ICAM-1 expression in normoxic epithelia, and
this response was lost in cells pre-exposed to ambient hypoxia. Such
results indicate that HIF-1-dependent down-regulation of PPAR- may
provide an adaptive response to proinflammatory stimuli during cellular
hypoxia. These studies provide unique insight into the regulation of
PPAR- expression and, importantly, provide an example of a
down-regulatory pathway mediated by HIF-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
(
), and ), each of which is encoded
by a different gene, has unique tissue distribution patterns, and each
has ligand-selective binding (1, 2). Recent studies have
identified a role for PPARs in the control of inflammation. For
example, PPAR- ligands were recently shown to inhibit NF-
B
activation and to diminish colonic inflammation in a mouse model of
inflammatory bowel disease (3). Similarly, ligands which
bind the PPAR- isoform may amplify or inhibit the expression of
inflammation-related gene products such as cyclooxygenase 2 and IL-6
(4, 5, 6) and may regulate the duration of inflammatory
responses through feedback pathways involving leukotriene
B4 (7).
In many disease states, hypoxia and inflammation occur coincidentally
(8). A number of studies have indicated that tissue
hypoxia associated with ongoing disease processes may amplify
proinflammatory signals and, paradoxically, may significantly
contribute to the resolution of ongoing inflammation (8).
For this reason, it is imperative to understand how induction pathways
of inflammation and hypoxia overlap. In addition, recent studies
indicate that hypoxia can directly activate transcription. This latter
response is exemplified by discovery of hypoxia-inducible factor 1
(HIF-1), a member of the rapidly growing Per-ARNT-Sim family of basic
helix-loop-helix transcription factors (9, 10). Functional
HIF-1 exists as an heterodimer, the activation of which is
dependent upon stabilization of an O2-dependent
degradation domain of the subunit by the ubiquitin-proteasome
pathway (11). Binding of HIF-1 to DNA consensus domains
(5'-RCGTG-3') results in the transcriptional induction of HIF-1-bearing
gene promoters (12). HIF-1 is widely expressed and recent
studies indicate that consensus HIF-1-binding sequences exist in a
number of genes (12). Transcriptional responses mediated
by HIF-1 include those ascribed to a hypoxia-adaptive response (e.g.,
erythropoietin, vascular endothelial growth factor, glycolytic enzymes,
etc.) (13). Less is known as to whether HIF-1 may function
to directly regulate inflammatory events or whether HIF-1 may mediate
negative (down-regulatory) pathways.
In the present studies, we identified a hypoxia-elicited
down-regulation of PPAR- in intestinal epithelial cells. In
parallel, these results identified a previously unappreciated binding
site for HIF-1 on the antisense strand of the PPAR- gene.
Down-regulation of PPAR- by hypoxia correlated with HIF-1
induction and functioned to protect epithelia from PPAR- agonist
amplification of ICAM-1 induction. These studies are the first to
define a down-regulatory pathway mediated by HIF-1 binding.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T84 intestinal epithelial cells (passages 67–85) were grown and maintained as confluent monolayers on collagen-coated permeable supports as previously described in detail (14). T84 monolayers were maintained on 0.33-cm2 or 5-cm2 ring-supported polycarbonate filters (Costar, Cambridge, MA), unless otherwise noted, and used 6–12 days after plating. The oral epithelial line (KB cells) were grown as described previously (15), and human microvascular endothelial cells were plated and cultured as described previously (16).
Monolayer exposure to hypoxia was performed as previously described (17). Growth medium was replaced with fresh, pre-equilibrated hypoxic medium and cells were placed in a humidified environment within the hypoxia chamber (Coy Laboratory Products, Ann Arbor, MI) and maintained at 37°C. Oxygen concentrations were maintained as indicated with the balance made up of nitrogen, 5% carbon dioxide, and water vapor. Normoxic controls were cells exposed to the same experimental protocols under conditions of atmospheric oxygen concentrations (21% O2/5% CO2 within a tissue culture incubator). Lactate dehydrogenase release into soluble supernatants was measured colorimetrically (Cytotox 96 Non-Radioactive Cytotoxicity Assay; Promega) according to the manufacturer’s protocol. Total cellular lactate dehydrogenase was determined from samples lysed in 0.5% Triton X-100.
Transcriptional analysis
The transcriptional profile of epithelial cells exposed to
ambient hypoxia was assessed in RNA derived from control or hypoxic
epithelia (T84 cells at 6 or 18 h hypoxia) using quantitative
genechip expression arrays (Affymetrix, Santa Clara, CA)
(18). RT-PCR analysis of mRNA levels was performed using
DNase-treated total RNA as previously described (19).
Briefly, single-stranded cDNA was synthesized from 1 µg of RNA (DNA
Polymerase High Fidelity PCR System; Life Technologies, Grand Island,
NY). The PCR for human PPAR- contained 1 µM each of the sense
primer (5'-TCA TCA AGA AGA CGG AGT CG-3') and the antisense primer
(5'-CGG TTA CCT ACA GCT CAG AC-3') in a total volume of 50 µl,
resulting in a 211-bp fragment, and for PPAR- (sense, 5'-AGA CAA CAG
ACA AAT CAC CAT-3' and antisense, 5'-AAG TTT GAG TTT GCT GTG AAG-3'),
resulting in a 401-bp fragment. PCR were then visualized on a 1%
agarose gel containing 5 µg/ml ethidium bromide. Human and mouse
-actin expression was examined in identical conditions as an
internal control (sense primer, 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT
CTA-3' and antisense primer, 5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA
GGG-3') revealing a 661-bp amplified fragment. Where indicated,
HIF-1 mRNA was examined by RT-PCR (sense primer, 5'-CTC AAA GTC GGA
CAG CCT CA-3' and antisense primer, 5'- CCC TGC AGT AGG TTT CTG CT
-3'), revealing a 460-bp amplified fragment.
Generation of nuclear lysates
For analysis of nuclear extracts, confluent monolayers of T84
cells on 100-mm petri dishes were washed in ice-cold PBS, lysed by
incubation in 500 µl of buffer A (10 mM HEPES (pH 8.0), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, 200 mM sucrose, 0.5
mM PMSF, 1 µg of both leupeptin and aprotinin per ml, and 0.5%
Nonidet P-40) for 5 min at 4°C. The crude nuclei released by lysis
were collected by microcentrifugation (15 s). Nuclei were rinsed once
in buffer A and resuspended in 100 µl of buffer C (20 mM HEPES (pH
7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5
mM PMSF, 1.0 mM DTT, and 1 µg/ml of both leupeptin and aprotinin).
Nuclei were incubated on a rocking platform at 4°C for 30 min and
clarified by microcentrifugation for 5 min. Proteins were measured
(detergent-compatible-protein assay; Bio-Rad, Hercules, CA).
Samples (25 µg/lane, as indicated) of T84 cell lysates were separated
by nonreducing SDS-PAGE, transferred to nitrocellulose, and blocked
overnight in blocking buffer (250 mM NaCl, 0.02% Tween 20, 5% goat
serum, and 3% BSA). For Western blotting, anti-HIF-1 (rabbit
antipeptide polyclonal directed to sequence MVNEFKLELVEKLFA encoding aa
527 through 541 of HIF-1) (20), anti-PPAR-
(Research Diagnostics, Flanders, NJ), or anti--actin (Santa Cruz
Biotechnology, Santa Cruz, CA) was added for 3 h, blots were
washed, and species-matched peroxidase-conjugated secondary Ab was
added exactly as described previously (27). Labeled bands
from washed blots were detected by ECL. Resulting bands were quantified
from scanned images using NIH Image software (Bethesda, MD).
EMSA
Nuclear extracts of cells exposed to indicated experimental
conditions were obtained as described above. The following synthetic
oligonucleotide probes were synthesized (Sigma-Genosys, The Woodlands,
TX) and used as probes in EMSAs; the hypoxic response enhancer
(HRE)-like motif (bold) on the antisense strand at positions 832–836
relative to the transcription start site in the PPAR- gene (sense,
5'-CTG CCA GTG CAC GTC AGT GGA G-3' and antisense, 5'-GAC
GGT CAC GTG CAG TCA CCT C-3'). Oligonucleotide probes for
EMSA were digoxigenin-labeled according to the manufacturer’s
instructions (gel shift kit; Boehringer Mannheim, Indianapolis, IN).
Labeled oligonucleotides were incubated with nuclear lysates for 10 min
at 37°C and separated by electrophoresis on a 6% nondenaturing
polyacrylamide gel. DNA-protein complexes were transblotted to nylon
membranes, probed with antidigoxigenin-peroxidase, and developed by
ECL. For supershift analysis, protein-DNA complexes were incubated with
anti-HIF-1 mAb (Transduction Laboratories, Lexington, KY) for
1 h at 4°C before electrophoresis. Controls consisted of free
probe alone, excess unlabeled probe, and isotype-matched control Ab
(anti-cAMP response element binding protein (CREB)-2; Santa Cruz
Biotechnology).
HIF-1 antisense oligonucleotide treatment of epithelia
HIF-1 depletion in epithelial cells was accomplished by using
antisense oligonucleotide loading as described previously
(21). Antisense oligonucleotide treatment of subconfluent
epithelial cells was done as described previously (21),
with modification. T84 epithelial cells were washed in serum-free
medium and then in medium containing 20 µg/ml Geneporter transfection
reagent (Gene Therapy Systems, San Diego, CA) with 2 µg/ml HIF-1
antisense or sense oligonucleotide. Cells were incubated for 4 h
at 37°C, then replaced with serum containing growth medium. Treated
cells were exposed to hypoxia or normoxia for indicated periods of
time. As indicated, PPAR- or HIF-1 mRNA were quantified by RT-PCR
as described above (see Transcriptional analysis).
ICAM-1 surface ELISA
IFN--induced ICAM-1 cell surface expression was quantified
using a cell surface ELISA as described before (22).
Epithelial cells were grown and assayed for Ab binding following
exposure to normoxia or hypoxia (24 h) in the presence or absence of
IFN- and addition of pirinixic acid (WY14643; Chemsyn Science
Laboratories, Lenexa, KS) for an additional 48 h as indicated.
Cells were washed with HBSS (Sigma, St. Louis, MO), and blocked with
medium for 30 min at 4°C. Anti-ICAM-1 mAb (clone P2A4
(23) obtained from the Developmental Studies Hybridoma
Bank, Iowa City, IA) was added for 2 h at 4°C. After washing
with HBSS, a peroxidase-conjugated sheep anti-mouse secondary Ab
(Cappel, West Chester, PA) was added. Secondary Ab (1:1000 final
dilution) was diluted in medium containing 10% FBS. After washing,
plates were developed by addition of peroxidase substrate
(2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 1 mM final
concentration; Sigma) and read on a microtiter plate spectrophotometer
at 405 nm (Molecular Devices, Menlo Park, CA). Controls consisted of
medium only and secondary Ab only. Data are presented as the mean
± SEM OD at 405 nm (background subtracted).
Mouse hypoxia model in vivo
Six- to 8-wk-old wild-type Bl6/129 (Taconic Farms, Germantown,
NY) were exposed to hypoxia (constant flow of 8%
O2, 92% N2 in a sealed
modular chamber) or ambient room air for 8 h (n =
3 per condition). At the end of the experiments, animals were
immediately sacrificed and tissues were collected for mRNA or protein
analysis. Human PPAR- PCR primers (both forward and reverse,
corresponding to nts 1550–1759 of mouse RNA) were used for mRNA
analysis, see Transcriptional analysis above).
This protocol was in accordance with National Institutes of Health
guidelines for use of live animals and was approved by the
Institutional Animal Care and Use Committee at Brigham and Women’s
Hospital and Harvard Medical School.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA
A transcriptional profiling approach was used to identify
potential hypoxia-regulated gene expression in model epithelia (T84
cells). As shown in Fig. 1
A,
microarray analysis identified a time-dependent down-regulation of the
PPAR- gene (2.1- and 8.9-fold decrease compared with control
normoxia at 6- and 18-h hypoxia, respectively). Such analysis suggested
this loss to be specific for PPAR-, since no apparent changes were
evident with PPAR- expression (0.05-fold loss and 0.1-fold increase
at 6- and 18-h hypoxia, respectively) and by microarray, PPAR- was
not expressed in model epithelia (data not shown). As shown in Fig. 1B, RT-PCR analysis was used to verify these microarray
results and revealed a time-dependent loss of PPAR-, but not
PPAR-, mRNA expression in hypoxia. Similar results of PPAR-, but
not PPAR-, loss were observed in an oral epithelial cell line (KB
cells, 92 ± 7% loss of PPAR- mRNA at the 6-h period of
hypoxia, n = 3, p < 0.01) and in
endothelial cells (human dermal microvascular endothelia, 65 ±
10% loss of PPAR- mRNA at the 8-h period of hypoxia,
n = 3, p < 0.05), suggesting that
these observations are not specific for intestinal epithelia.
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is HIF-1 dependent
Prompted by these results, a search of the PPAR- gene sequence
identified a previously unappreciated HIF-1 binding site at
positions 832–836 (relative to the first methionine codon) on the
antisense strand of PPAR- (DNA consensus motif 5'-ACGTG-3')
(24). Consequently, we assessed the induction of HIF-1
and loss of PPAR- protein during subjection of epithelia to ambient
hypoxia. First, we determined whether conditions of hypoxia induce
HIF-1 in intestinal epithelia (Fig. 2A). Western blot analysis of
nuclear lysates derived from hypoxic intestinal epithelia demonstrated
detectable HIF-1 at 2 h and abundant HIF-1 by 4 h of
hypoxia. -Actin was used as a loading control for these experiments.
These data are consistent with previous studies suggesting that
HIF-1 is expressed in most cell types during periods of hypoxia and
localizes to the nucleus during hypoxia exposure (25).
Parallel examination of PPAR- protein by Western blot revealed a
time-dependent loss over the course of 12 h of exposure to hypoxia
(by densitometry, 63 and 84% loss at 8- and 12-h hypoxia,
respectively, compared with control). Exposure of normoxic epithelial
cells to CoCl2 (100 µM, 8 h), a
well-recognized activator of HIF-1 (9), also resulted
in a loss of PPAR- protein (72% decrease by densitometry),
suggesting that other known activators of HIF-1 similarly diminish
PPAR-. -Actin was used as a loading control for these
experiments. Such data indicate a temporal induction of HIF-1 and loss
of PPAR- protein during hypoxia.
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to the putative HRE consensus
(Fig. 3A) in the PPAR- gene
by EMSA. As shown in Fig. 3B, nuclear extracts derived from
4- or 24-h hypoxic, but not normoxic, epithelial cells bind to the
HIF-1 consensus of the PPAR- gene. Pretreatment of epithelia with
CoCl2 (100 µM, 4 h), conditions which
promote nuclear accumulation of HIF-1 (10), resulted in
a similar band shift. Addition of anti-HIF-1 mAb, but not
anti-CREB-2, resulted in an obvious super shift indicative of
HIF-1 binding to this oligonucleotide. Incubation with 100-fold
excess unlabeled primer resulted in complete loss of signal.
Collectively, these results indicate that hypoxia elicits nuclear
accumulation of HIF-1 which binds to the consensus HRE located
within the PPAR- gene.
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was depleted in
intestinal epithelial cells using antisense oligonucleotides (Fig. 4A). Such treatment with
antisense, but not sense oligonucleotides, resulted in a complete loss
of HIF-1 mRNA, consistent with previous studies using these
oligonucleotides (21). Cells treated in this manner were
subjected to hypoxia and examined for the loss of PPAR- mRNA (Fig. 4C) and protein (Fig. 4D). As can be seen, the
absence of HIF-1 resulted in a diminished loss of PPAR- during
hypoxia. Control cells exposed to sense oligonucleotides remained
responsive to hypoxia with the observed down-regulation of PPAR-
mRNA and protein. These data provide additional evidence for HIF-1 in
the loss of PPAR- during hypoxia.
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in hypoxia
Given the findings described above, studies were undertaken to
determine whether PPAR- was functional in epithelial cells and
whether such functional responses were lost during hypoxia. Previous
studies have suggested that the PPAR- ligands such as pirixinic acid
(WY14643) may enhance proinflammatory gene expression in epithelial
cells (4, 5). Thus, as a functional assay for PPAR-
activation, we examined the induction of epithelial ICAM-1 by IFN-
(22, 26) in the presence and absence of PPAR activators.
As shown in Fig. 5, the PPAR- ligand
pirinixic acid enhanced IFN--induced ICAM-1 expression in epithelial
cells (Fig. 5, p < 0.025 by ANOVA). Consistent
with previous studies (22, 26), ICAM-1 was not expressed
to an appreciable amount in the absence of IFN-. Concentrations as
low as 500 nM pirinixic acid significantly enhanced IFN--induced
ICAM-1 induction (Fig. 5, p < 0.025). Similar
experiments performed on epithelial cells pre-exposed to 24-h hypoxia
revealed a loss of pirixinic acid influence (p
= NS by ANOVA). Such findings indicate that PPAR- is functional in
epithelial cells, and consistent with our biochemical evidence, hypoxia
induces a loss of functional PPAR- in the setting of an inflammatory
stimulus (IFN-).
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in vivo
We next evaluated whether hypoxia elicits the loss of PPAR- in
vivo. Similar to the human PPAR- gene HIF-1 binding site, the mouse
PPAR- gene includes an identical HIF-1-binding motif on the
antisense strand (DNA consensus 5'-ACGTG-3' located at positions
905–909 relative to the first methionine codon) as well as identical
flanking regions around this site (27). Since PPAR- is
expressed at high levels in mouse intestine (28) and our
results using cultured intestinal epithelia (T84 cells) revealed a loss
of PPAR-, we compared PPAR- mRNA and protein levels in intestinal
tissue following mouse subjection to whole-body hypoxia (8%
O2, 92% N2 for 8 h)
or to ambient room air. A similar hypoxia model has been used to
examine hypoxia-regulated gene products in a variety of tissues
(29, 30, 31). As shown in Fig. 6, lumenal scrapings (enriched in
epithelial cells) harvested from intestinal tissue derived from hypoxic
mice revealed a significant decrease in both PPAR- mRNA (93 ±
17% decrease compared with normoxic control, n = 3
mice per condition, p < 0.01) and protein (71 ±
12% decrease compared with normoxic control, n = 3 per
condition, p < 0.025). These data indicate the
likelihood that similar HIF-1-mediated down-regulatory pathways exist
in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression. In parallel, decreased protein expression manifests as a
loss PPAR- function. Initial mechanistic insight was gained through
identification of a putative HRE in the PPAR- gene, and significant
evidence defines a dominant role for HIF-1 in this response. Taken
together, these data provide an example of HIF-1-mediated
down-regulation of gene expression.
Previous studies have demonstrated that PPARs are multifunctional
nuclear receptors which when activated by a variety of fatty acids and
prostaglandins regulate gene transcription via localization to
recognition sequences termed peroxisome proliferator response elements
(PPREs) (2). It is unclear, however, how PPAR genes are
transcriptionally regulated. In the present studies, several lines of
evidence identify a HIF-1-mediated repressor activity for PPAR-.
Initial insight was gained through a broad screen of epithelial genes
by microarray analysis, a strategy that has proven beneficial for
identifying hypoxia-regulated pathways in epithelial cells (32, 33). Subsequent studies identified a specificity for such
responses (e.g., not evident for PPAR-), some degree of
universality for this response (i.e., also apparent in oral epithelial
cell line KB and in human microvascular endothelial cells), and the
existence of a putative hypoxia response enhancer within the PPAR-
gene. To date, defined HIF-1 regulatory pathways have been limited to
induction of hypoxia-regulated genes (34), and we provide
an example here of HIF-1-mediated repressor activity. Evidence for this
HIF-1-mediated pathway includes 1) the existence of a consensus binding
site for HIF-1, 2) recapitulation of this response with a known
activator of HIF-1 (CoCl2), 3) HIF-1 binding by
EMSA, and 4) inhibition of this response in cells not expressing
HIF-1 (antisense oligonucleotides).
It is not known how HIF-1 repressor activity manifests itself. It is
possible that binding of the HIF-1 heterodimer to the antisense
strand of some genes (e.g., PPAR-) is directional and, as such,
results in a functional transcriptional repressor. This aspect of HIF-1
signaling has not been studied in detail. Alternatively, HIF-1 may
associate with other hypoxia-modified transcriptional regulators to
maintain PPAR- expression. For example, we have recently
demonstrated that CREB is targeted for degradation under conditions of
hypoxia (19, 33). Given that the human PPAR- gene
sequence includes a previously unappreciated CREB binding site (DNA
consensus 5'-TGACGGA-3' at positions 1309–1315 relative to the
translation start site) (24) downstream from the HIF-1
binding site (nt positions 832–836) and HIF-1 DNA binding sites have
been shown to interact with CREB protein (35, 36), a
potential pathway could involve destabilization of PPAR- expression
through the loss of CREB. Thus, details of PPAR- down-regulation by
HIF-1 remain unclear and will require extensive additional studies.
HIF-1 is particularly interesting as a regulatory pathway for mucosal
transcriptional responses. First, mucosal organs, such as the lung and
intestine, support a rich underlying vasculature, and perturbations in
blood flow can result in rapid and severe tissue hypoxia
(8). Important in this regard, epithelial cells that line
mucosal tissues are active participants in the inflammatory response,
and hypoxia may contribute substantially to such ongoing inflammation
(8). We demonstrate here that PPAR--selective ligands,
such as pirixinic acid, enhance epithelial ICAM-1 induction, an
inflammatory marker on intestinal epithelial cells in vitro and in vivo
(22). These findings of enhanced ICAM-1 induction are
consistent with one study in endothelial cells (37),
although other evidence suggests that PPAR ligands may inhibit
TNF-induced ICAM-1 expression (38). Our own analysis of
the published 5' region of the ICAM-1 gene (39)
identified a PPAR response element (PPRE)-like consensus sequence
(2) (consensus AGGTCATGCATGCTTAGGT
positioned 197–215 nts downstream of the TATA signal), providing at
least the possibility that the ICAM-1 gene directly binds
PPAR. Detailed studies are necessary to further define the identity of
PPRE within the ICAM-1 gene. Alternatively, it is possible
that PPAR- interacts with other inflammatory pathways (e.g.,
NF-B, mitogen-activated protein kinases) and that PPAR-
activity on ICAM-1 is indirect, particularly since the activity of the
PPAR- ligand required additional inflammatory stimuli (i.e.,
IFN-). Nonetheless, our data that PPAR- ligand-mediated induction
of proinflammatory signals (e.g., ICAM-1) are consistent with previous
observations that PPAR- ligands enhance cyclooxygenase 2 in colonic
and corneal epithelial cells (4, 5), and such observations
may indicate that PPAR- provides a proinflammatory signal to
epithelial cells. Of note, the isoform of PPAR, which was not
regulated by hypoxia (see Fig. 1
), has recently been targeted as an
anti-inflammatory therapeutic pathway for mucosal disorders such as
Crohn’s disease (3). Second, it is possible that
HIF-1-mediated PPAR- expression could regulate epithelial cancers,
such as colon cancer. For instance, recent studies have indicated that
induction of cyclooxygenase 2 may be rate limiting to the development
of colon cancer (40, 41, 42), and that cyclooxygenase
inhibitors may afford some protection in this regard (43).
Along these same lines, a mounting body of evidence has demonstrated
that HIF-1-mediated genes are crucial to the establishment and
maintenance of solid tumors (44, 45). Our studies with
PPAR- may provide additional insight into these pathways. Taken
together, these results of HIF-1-mediated down-regulation of PPAR-
may define a novel adaptive pathway to dampen mucosal inflammation and
limit epithelial proliferation responses.
In summary, these results define a new pathway of PPAR-
transcriptional regulation. Such results indicate that
pathophysiologically relevant conditions (e.g., hypoxia) have the
potential to directly impact PPAR signaling through regulation of the
nuclear receptor. Moreover, these studies provide an example of a
counterregulatory transcriptional pathway for HIF-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sean P. Colgan, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Thorn Building, Room 704, 20 Shattuck Street, Boston, MA 02115. E-mail address: colgan{at}zeus.bwh.harvard.edu
3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; HIF-1, hypoxia-inducible factor 1; CREB, cAMP response element binding protein; HRE, hypoxic response enhancer; PPRE, peroxisome proliferator response element.
Received for publication November 20, 2000. Accepted for publication April 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 H. R. Gosker, P. Schrauwen, R. Broekhuizen, M. K. C. Hesselink, E. Moonen-Kornips, K. A. Ward, F. M. E. Franssen, E. F. M. Wouters, and A. M. W. J. Schols Exercise training restores uncoupling protein-3 content in limb muscles of patients with chronic obstructive pulmonary disease Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E976 - E981. [Abstract] [Full Text] [PDF]
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 U. Flogel, T. Laussmann, A. Godecke, N. Abanador, M. Schafers, C. D. Fingas, S. Metzger, B. Levkau, C. Jacoby, and J. Schrader Lack of Myoglobin Causes a Switch in Cardiac Substrate Selection Circ. Res., April 29, 2005; 96(8): e68 - e75. [Abstract] [Full Text] [PDF]
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T. A. Gorr, J. D. Cahn, H. Yamagata, and H. F. Bunn Hypoxia-induced Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent J. Biol. Chem., August 20, 2004; 279(34): 36038 - 36047. [Abstract] [Full Text] [PDF]
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T. A. Gorr, T. Tomita, P. Wappner, and H. F. Bunn Regulation of Drosophila Hypoxia-inducible Factor (HIF) Activity in SL2 Cells: IDENTIFICATION OF A HYPOXIA-INDUCED VARIANT ISOFORM OF THE HIF{alpha} HOMOLOG GENE similar J. Biol. Chem., August 20, 2004; 279(34): 36048 - 36058. [Abstract] [Full Text] [PDF]
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N. Wakisaka, S. Kondo, T. Yoshizaki, S. Murono, M. Furukawa, and J. S. Pagano Epstein-Barr Virus Latent Membrane Protein 1 Induces Synthesis of Hypoxia-Inducible Factor 1{alpha} Mol. Cell. Biol., June 15, 2004; 24(12): 5223 - 5234. [Abstract] [Full Text] [PDF]
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E. S. Tien, J. W. Davis, and J. P. Vanden Heuvel Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor {alpha} Coregulator J. Biol. Chem., June 4, 2004; 279(23): 24053 - 24063. [Abstract] [Full Text] [PDF]
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J. T. Erler, C. J. Cawthorne, K. J. Williams, M. Koritzinsky, B. G. Wouters, C. Wilson, C. Miller, C. Demonacos, I. J. Stratford, and C. Dive Hypoxia-Mediated Down-Regulation of Bid and Bax in Tumors Occurs via Hypoxia-Inducible Factor 1-Dependent and -Independent Mechanisms and Contributes to Drug Resistance Mol. Cell. Biol., April 1, 2004; 24(7): 2875 - 2889. [Abstract] [Full Text] [PDF]
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S. Yim, S. M. Choi, Y. Choi, N. Lee, J. Chung, and H. Park Insulin and Hypoxia Share Common Target Genes but Not the Hypoxia-inducible Factor-1{alpha} J. Biol. Chem., October 3, 2003; 278(40): 38260 - 38268. [Abstract] [Full Text] [PDF]
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L. Ulloa, M. Ochani, H. Yang, M. Tanovic, D. Halperin, R. Yang, C. J. Czura, M. P. Fink, and K. J. Tracey Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation PNAS, September 17, 2002; 99(19): 12351 - 12356. [Abstract] [Full Text] [PDF]
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 I. Pineda Torra, Y. Jamshidi, D. M. Flavell, J.-C. Fruchart, and B. Staels Characterization of the Human PPAR{alpha} Promoter: Identification of a Functional Nuclear Receptor Response Element Mol. Endocrinol., May 1, 2002; 16(5): 1013 - 1028. [Abstract] [Full Text] [PDF]
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) or hypoxia (
) for 48 h,
after which ICAM-1 expression was assessed by cell surface ELISA. Data
are pooled from three separate experiments and presented as the
mean ± SEM ICAM-1 expression.