1 Department of Cell Biology, Faculty of Biology, University of Kaiserslautern, 67663 Kaiserslautern; 2 Institute of Physiology, Faculty of Medicine, University of Essen, 45122 Essen; and 3 Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Erlangen, 91054 Erlangen, Germany
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ABSTRACT |
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Hypoxic/ischemic conditions
provoke activation of the transcription factor hypoxia-inducible
factor-1 (HIF-1). HIF-1 is composed of HIF-1 (subjected to protein
stability regulation) and constitutively expressed HIF-1
. Besides
hypoxia, diverse agonists are identified that stabilize HIF-1
during
normoxia. Here we used a coculture system of RAW 264.7 macrophage cells
and tubular LLC-PK1 cells to establish that
lipopolysaccharide- and interferon-
-stimulated but not resting
macrophages elicited HIF-1
accumulation in LLC-PK1 cells. Via pharmacological interventions such as blockade of nitric oxide (NO) production in macrophages, scavenging of NO with the use of
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, or application
of tumor necrosis factor-
(TNF-
)-neutralizing antibodies, we
identified NO and TNF-
as signaling molecules. Working in concert,
NO and TNF-
have a stronger response when allowed direct
cell-to-cell contact instead of contact with only the cell supernatant
of activated macrophages. We show that signal transmission by NO with
TNF-
in LLC-PK1 cells is mediated via the
phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, because it is
blocked by wortmannin or dominant-negative forms of PI3-K as well as
protein kinase B. We conclude that NO and TNF-
, derived from
activated macrophages, provoke HIF-1
stabilization in
LLC-PK1 cells under normoxic conditions, which underscores
HIF-1
stabilization due to intercellular regulation.
nitric oxide; tumor necrosis factor-; hypoxia-inducible
factor-1; intercellular signaling; phosphatidylinositol 3-kinase; cytokine; Akt
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INTRODUCTION |
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HYPOXIA-INDUCIBLE
FACTOR-1 (HIF-1), a hypoxic inducible transcription
factor complex, is a heterodimer that is composed of the basic
helix-loop-helix PER-ARNT-SIM proteins HIF-1 and the aryl
hydrocarbon nuclear translocator that is also known as HIF-1
(9, 34, 35). An active HIF-1 heterodimer binds to the
HIF-1 binding site within the hypoxia-response element and enhances transcription of hypoxia-inducible genes involved in glucose/energy metabolism, cell proliferation and viability, erythropoiesis, iron
metabolism, vascular development, or remodeling. Moreover, it is
appreciated that a hypoxic environment in tumors is associated with
angiogenesis, and expression of HIF-1 activity promotes tumor growth,
vascularization, and energy metabolism (14).
Overexpression of HIF-1
is found in common cancer, which is a
consequence of protein stabilization achieved by various agonists or
genetic alterations. Resistance of hypoxic tumor cells to killing by
radiation or chemotherapy could be due in part to the induction of
HIF-1
(17, 37).
The availability of HIF-1 is predominantly determined by stability
regulation of HIF-1 via proline hydroxylation (19, 20, 39). HIF-1
is degraded under normoxic conditions. Studies in von Hippel-Lindau protein (pVHL)-defective renal cell carcinomas (27) indicated a critical role for pVHL in HIF-1
degradation. Recent evidence suggests that proly hydroxylases (HIF-PH)
sense oxygen and function as putative oxygen sensors. These enzymes target highly conserved proline residues at HIF-1
positions 564 and/or 402 to hydroxylate HIF-1
(19, 20). Proline
hydroxylation appears to be necessary and sufficient for the binding of
pVHL to HIF-1
with concomitant degradation of HIF-1
by the
ubiquitin-proteasome system. Besides hypoxia, HIF-1 can be activated by
transition metals and by reagents that chelate iron such as
desferroxamine, which is often chosen to mimic hypoxia
(35). For these compounds and hypoxia, it is rationalized
that direct inhibition of HIF-PH accounts for HIF-1
stabilization
(36).
Among recent advances in the understanding of oxygen sensing are the
discoveries that reactive oxygen species (ROS), nitric oxide (NO),
carbon monoxide, cytokines, and growth factors participate in
regulation of HIF-1 during normoxia (35). Regulation of
HIF-1 activity by NO is likely to be of pathophysiological relevance, but details at this point are not clear. Initial observations suggested
that NO inhibits hypoxia-induced HIF-1 stabilization and HIF-1
transcriptional activation (18, 25, 38). Later studies
indicated that chemically diverse NO donors or endogenous NO formation
under normoxic conditions induced HIF-1
stabilization, HIF-1 DNA
binding, and activation of downstream target-gene expression (see Refs.
8, 12, 22, 29,
31). For ROS, the situation is complex as well. There is
experimental evidence in support of the hypothesis that mitochondrial
generation of superoxide and dismutation to
H2O2 are required for induction of HIF-1
activity and target-gene activation, which thereby implies ROS
generation under hypoxia. An alternative model proposes that hypoxia
decreases production of ROS and that mitochondria-derived oxygen
species are not involved in regulation of HIF-1
stability (34,
35). Reports on cytokines in HIF-1
stability regulation have
indicated that interleukin-1
(IL-1
) and tumor necrosis factor-
(TNF-
) are positively involved (16, 32, 40). The term
stabilization refers to a situation whereby the HIF-1
protein
accumulates, which implies inhibition of proline hydroxylation.
However, recent data (24, 37) show that the stability of
HIF could also be regulated at the translational level. Furthermore, it
seems that stabilization and transactivation of HIF-1
are two
separate processes that are regulated by hydroxylation at distinct
residues, i.e., proline 564 and asparagine 803 (23, 37).
With regard to intracellular signal transmission in the regulation of
HIF-1, activation of the phosphatidylinositol 3-kinase (PI3-K),
serine/threonine kinases [protein kinase B (PKB)/Akt], FK506 binding
protein (FKBP)-rapamycin-associated protein (FRAP), or
mitogen-activated protein kinases (MAPK) are implicated (27, 35). Even though contradictory data have shown that PI3-K/Akt signaling is cell-type specific and this pathway may not be sufficient to induce HIF transactivation during hypoxia (2, 4), one may envision the involvement of these pathways under normoxic situations by distinct agonists. Herein, we tested the hypothesis that
intercelluar signal communication may affect HIF-1 stability regulation. We provide evidence by using a coculture setup whereby activated macrophages release NO and TNF-
as soluble messengers that
provoke an HIF-1
response in resting tubular LLC-PK1
cells called detector cells. We conclude that an HIF-1 response may be
found in close association with inflammatory conditions, because it is
elicited by proinflammatory molecules that signal between different
cell types.
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MATERIALS AND METHODS |
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Materials.
Medium and supplements were purchased from Biochrom, and fetal calf
serum was bought from Life Technologies (both from Berlin, Germany).
Nitrocellulose and the enhanced chemiluminescence (ECL) detection
system came from Amersham (Freiburg, Germany). HIF-1 and p85
antibodies were ordered from Becton Dickinson (Heidelberg, Germany),
the Akt antibody came from New England Biolabs (Frankfurt, Germany),
and secondary antibodies were delivered by Promega (Mannheim, Germany).
Wortmannin, diethylenetriamine-nitric oxide (DETA-NO), and anti-actin
antibody were ordered from Sigma (Deisenhofen, Germany). All other
chemicals were of the highest grade of purity and were commercially
available. Plasmids pSR
-
p85 and pSR
-WTp85 were kindly provided
by Dr. W. Ogawa (School of Medicine, Kobe University, Kobe, Japan; see
Ref. 15). Plasmids pCMV5 and pCMV5.-m/p-PKB
K179 were a
gift from Dr. B. Hemmings (F. Miescher Institute, Basel, Switzerland).
Cell culture. Proximal tubular porcine LLC-PK1 cells (obtained from Prof. D. Dietrich, Konstanz, Germany) and/or RAW 264.7 macrophages were cultured in DMEM with 1 g/l glucose supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum. Cells were transferred two times each week, and medium was changed before experiments. Cells were kept in a humidified atmosphere of 5% CO2 in air at 37°C.
Coculture of LLC-PK1 and RAW 264.7 cells.
LLC-PK1 cells (4 × 105) were seeded in a
6-cm dish and RAW 264.7 cells (1 × 107) were
cultured in 10-cm dishes. RAW 264.7 cells were stimulated with
lipopolysaccharide (LPS, 1 µg/ml) and interferon- (INF-
, 100 U/ml) for 18 h. After stimulation, RAW 264.7 cells were washed with PBS, scraped from the dish, and centrifuged at 500 g
for 5 min. Where indicated, the supernatant of activated macrophages was used to replace the medium of LLC-PK1 cells. If the
anti-TNF-
antibody was supplied, the supernatant of activated
macrophages was incubated with the antibody (50 µg/ml) at 37°C for
10 min before it was added to the LLC-PK1 cells. RAW 264.7 cells were resuspended in culture medium, and 8 × 105
RAW 264.7 cells were cocultured with 4 × 105
LLC-PK1 cells for 8 h. When necessary,
LLC-PK1 cells were treated with 100 nM wortmannin for 30 min before RAW 264.7 cells were added.
Western blot analysis.
HIF-1, p85, or Akt were quantified by Western blot analyses.
Briefly, LLC-PK1 cells were incubated for the times
indicated, scraped off the dishes, lysed in 150 µl of lysis buffer
[50 mM Tris · HCl, 5 mM EDTA, 150 mM NaCl, 0.5%
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0], and
sonicated. After centrifugation at 17,000 g for 15 min, the
protein contents in the supernatants were analyzed. Finally, 100 µg
of protein were added to the same volume of 2× sample buffer [125 mM
Tris · HCl, 2% SDS, 10% glycerin, 10 mM
dithiothreitol (DTT), 0.002% bromphenol blue, pH 6.9] and boiled for
5 min. Proteins were resolved on 7.5% SDS-polyacrylamide gels and
blotted onto nitrocellulose. Molecular weights were calibrated in
proportion to the running distance of markers. Membranes were washed
twice with Tris-buffered saline (TBS; 140 mM NaCl and 50 mM
Tris · HCl, pH 7.2) that contained 0.1% Tween 20 before unspecific binding was blocked with TBS and 5% skim milk for
1 h. The HIF-1
antibody (1:250 dilution in TBS with 0.5%
milk), p85 antibody (1:2,500 dilution in TBS with 0.5% milk), or Akt
antibody (1:1,000 dilution in TBS with 0.5% milk) was added and
incubated overnight at 4°C. Afterward, nitrocellulose membranes were
washed five times for 15 min with TBS that contained 0.1% Tween 20. For protein detection, blots were incubated with goat anti-mouse
secondary antibodies or, in the case of Akt, with goat anti-rabbit
secondary antibodies conjugated with peroxidase (1:10,000 dilution in
TBS with 0.2% milk) for 45 min, which was followed by ECL detection.
Transfections. LLC-PK1 cells (4 × 105) were seeded in 6-cm dishes 1 day before transfection. After reaching 70% confluence, cells were transfected with 3 µg of the different plasmids. Therefore, 35 µl of a 5 mM polyethylenimine solution was mixed with 85 µl of medium without serum and 3 µg of the selected DNA. Mixtures were vortexed for 5 s, incubated for 30 min at room temperature, and added dropwise to the cells that contained 2.5 ml of medium with supplements. Medium was changed 4 h later, and cells were stimulated as indicated 24 h later.
ELISA-based TNF- determinations.
As previously described (33), detection of TNF-
in
culture supernatants was performed on flat-bottomed, high-binding,
polystyrene microtiter plates (Nunc, Roskilde, Denmark) using
specific monoclonal antibody pairs (anti-mouse TNF-
and biotinylated
anti-mouse TNF-
) and avidin-horseradish peroxidase
conjugate, which were purchased from PharMingen (San
Diego, CA).
Densitometric quantification and statistical analysis.
Densitometric quantification of Western blot signals, i.e., HIF-1
bands, was performed with Aida Image software (Raytest Isotopenmessgeraete). When densitometry is measured relative to the
appearance of actin, results are expressed as relative quantum levels
(QLs). Each experiment was performed at least three times, and
representative data are shown. Values are means ± SE.
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RESULTS |
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Activated macrophages provoke HIF-1 accumulation in
LLC-PK1 cells.
During inflammation, macrophages are activated and proinflammatory
mediators are released. To study HIF-1
accumulation during inflammation, we employed tubular LLC-PK1 cells that were
cocultured with murine RAW 264.7 macrophages. When 4 × 105 tubular LLC-PK1 cells were cocultured with
8 × 105 RAW 264.7 macrophages for 8 h, we
detected neither an HIF-1
signal on Western blots nor the NO
oxidation product nitrite in the cell supernatant. To simulate an
activated state, macrophages were stimulated for 18 h with a
combination of 1 µg/ml LPS and 100 U/ml INF-
. First, we ensured
that no HIF-1
signal emerged in resting or activated macrophages
(Fig. 1, lanes I-III).
Thereafter, activated macrophages were cocultured with resting
LLC-PK1 cells for an additional 8-h period. As shown in
Fig. 1, this situation evoked an HIF-1
response as well as nitrite
formation. Because RAW 264.7 cells did not stabilize HIF-1
in
response to LPS/IFN-
addition, it must be concluded that the
HIF-1
signal exclusively originated from the LLC-PK1
cells. Activation of macrophages was followed by nitrite formation that
amounted to 5.4 µM in the cell supernatant during 8 h of the
coculture of LLC-PK1 and RAW 264.7 cells. When the NO
synthase inhibitor L-NAME at a concentration of 1 mM was
added to activated macrophages during coculture, HIF-1
stabilization
in LLC-PK1 cells was largely attenuated. This correlates with the efficacy of L-NAME to block nitrite formation in
macrophages.
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Macrophages use multiple signals to stabilize HIF-1 in
LLC-PK1 cells.
In searching for HIF-1
-stabilizing factors that are released by
activated macrophages, we either cultured RAW 264.7 macrophages directly with LLC-PK1 cells or transferred the cell
supernatant of macrophages to only LLC-PK1 detector cells.
As depicted in Fig. 5A, when
cocultured for 8 h with LLC-PK1 cells, activated but
not resting macrophages caused HIF-1
stabilization.
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DISCUSSION |
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By coculturing macrophages and tubular LLC-PK1 cells,
we established that HIF-1 is subjected to stability regulation by
soluble intercellular messengers. NO and TNF-
released from
activated but not resting macrophages stabilized HIF-1
in
LLC-PK1 detector cells. Activation of intracellular
signaling pathways as a result of NO and TNF-
formation comprised
the PI3-K/Akt pathway, and signal strength demanded both messengers to
act in concert.
HIF-1 plays essential roles in mammalian development, physiology, and
disease pathogenesis. Although oxygen availability may influence
multiple steps in HIF-1 stability, regulation of the primary mode of
action occurs through oxygen-dependent proteolysis of HIF-1 subunits
that become stabilized during hypoxia (11, 26). Recent
advances pointed out that HIF-1 activity regulation is achieved by
signals other than hypoxia including NO, cytokines such as TNF-
,
growth factors, or hormones (1, 6, 8, 13, 30, 35, 42).
Although diverse factors stimulate HIF-1
accumulation and activation
in a variety of cell types, we still lack detailed knowledge about how
intercellular signaling mechanisms converge in HIF-1
stability
regulation and whether individual agonists combine effects. Most
likely, multiple signaling pathways are capable of modulating HIF-1
expression and/or activity either by altering prolyl hydroxylation,
modulating this process, or utilizing alternative direct and/or
indirect posttranslational modification mechanisms. Phosphorylation
cascades involving PI3-K, the serine/threonine kinase PKB/Akt, or the
p42/p44 MAPK cascade may aim at this direction.
To date, studies have primarily focused on intracellular singling
pathways that participate in HIF-1 stability regulation. Despite the
complex network of signaling mechanisms, most studies used hypoxia or a
receptor-triggered cascade and focused on the formation of
intracellular messengers that affect protein stability regulation of
HIF-1
. In our study, we proceeded to demonstrate accumulation of
HIF-1
due to intercellular signaling pathways. This opens the
possibilities of autocrine- and paracrine-stability regulation of
HIF-1
. Thus the HIF-1
-activating signal may be produced in a
bystander or (in terms of space) separate cell, which broadens the
repertoire of regulatory mechanisms of the HIF-1 response. HIF-1
regulation is now put into the complex network of intra- and
intercellular communication.
In our coculture system, activated macrophages provoked HIF-1
accumulation in LLC-PK1 detector cells. NO and TNF-
are
produced upon macrophage activation, and both factors are needed to
transmit the signal from macrophages to tubular cells. This is
confirmed by use of the specific pharmacological agent
L-NAME to block NO synthase, by employing the NO scavenger
c-PTIO, or by supplying TNF-
-neutralizing antibodies. Blocking
and/or eliminating either NO or TNF-
abrogated signal transmission
to a large extent. We must conclude that under coculture conditions,
only the combined biological activity of NO and TNF-
achieves
maximal HIF-1
accumulation. This is somewhat in analogy to the study
of Hellwig-Bürgel et al. (16), who found that
cytokines such as IL-1
or TNF-
strongly augmented HIF-1 activity
in hypoxic HepG2 cells compared with the effect of hypoxia alone, which
was not followed by protein accumulation. We confirmed stronger
HIF-1
protein accumulation when treating LLC-PK1 cells
with a combination of DETA-NO and TNF-
, although NO donors as well
as TNF-
are known to be self-sufficient in eliciting an HIF-1
response (1, 12, 13, 22, 31, 32). When elicited by
activated macrophages, HIF-1
stabilization in detector cells makes
use of established and known signaling pathways. However, blocking
either the NO or the TNF-
portion of the signal abrogated HIF-1
stabilization to a great extent. We must assume that the timing of the
signal output and the signal strength, i.e., the steady-state
concentrations of agonists, are more important than is appreciated in
experiments whereby a one-cell system and exogenously added HIF-1
inducers are used. In our experimental setup, which mimicks
endogenous/pathophysiological signal generation, only the combination
of at least two signaling pathways elicits a major HIF-1
response.
In the particular case of TNF- and NO, a receptor-mediated event and
a nonclassical signaling molecule combine to achieve signal
transmission. Despite the fact that NO signaling is cGMP independent
(29, 31), multiple intracellular targets are feasible, and
the impact of NO on gene activation and multiple transcription factors
is described (7). For NO, it is essential to define whether it attenuates prolyl hydroxylation by interaction with the
enzyme-bound iron or whether S-nitrosylation, nitration, oxidation, or
phosphorylation is encountered during signaling. Furthermore, we must
learn how these potential target interactions channel into a
PI3-K-sensitive pathway. TNF-
is reported to activate PI3-K and to
cause concomitant Akt phosphorylation (28) probably via
c-Src (5, 41). Therefore, it is without surprise that a
PI3-K inhibitor such as wortmannin, a dominant-negative PI3-K-isoform, or an inactive Akt kinase attenuates HIF-1
stabilization in response to TNF-
. Despite the contradiction on the role of PI3-K in HIF-1
stabilization during hypoxia, NO and TNF-
signals converge upstream of PI3-K activation, which is in some agreement with the importance of
the PI3-K pathways on basal-, growth factor-, and mitogen-induced expression of HIF-1
(10, 43). Future work needs to
address how a PI3-K signal manages to attenuate HIF-1
degradation
and whether activation of nuclear factor-
B or activator protein-1, two well-established downstream signaling pathways of TNF-
, are involved.
Considering NO and TNF- as important intercellular messengers that
provoke an HIF-1
response, it may be justified to put their
formation and action into a pathophysiological, i.e., inflammatory and/or tumorgenic, context. TNF-
is a potent proinflammatory cytokine, and NO can be considered a marker of inflammatory host defense. Formation of TNF-
and NO is essential for cell
proliferation, differentiation, and apoptosis, and thus
HIF-1
stabilization may contribute to the regulation of these vital
cell responses. Moreover, generation of NO in tissue is known to
promote angiogenesis (21), which may at least in part be
explained by NO-evoked HIF-1
stabilization. One may speculate
whether HIF-1 not only helps to adapt to conditions of reduced oxygen
availability but also functions in coordinating a proper cell response
to inflammation. Under these conditions, stability regulation of
HIF-1
by intercellular messengers makes perfect sense.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. W. Ogawa (Kobe University School of Medicine, Kobe, Japan) and Dr. B. Hemmings (F. Miescher Institute, Basel, Switzerland) for providing the p85 and PKB plasmids.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Grant BR999, European Community Grant QLK6-CT-2000-00064, and Fonds der Chemischen Industrie.
Address for reprint requests and other correspondence: B. Brüne, Dept. of Cell Biology, Faculty of Biology, Univ. of Kaiserslautern, Erwin-Schroedinger-Strasse, 67663 Kaiserslautern, Germany (E-mail: bruene{at}rhrk.uni-kl.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 25, 2002;10.1152/ajpcell.00294.2002
Received 25 June 2002; accepted in final form 20 September 2002.
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