NO and TNF-alpha released from activated macrophages stabilize HIF-1alpha in resting tubular LLC-PK1 cells

Jie Zhou1, Joachim Fandrey2, Jens Schümann3, Gisa Tiegs3, and Bernhard Brüne1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxic/ischemic conditions provoke activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is composed of HIF-1alpha (subjected to protein stability regulation) and constitutively expressed HIF-1beta . Besides hypoxia, diverse agonists are identified that stabilize HIF-1alpha 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-gamma -stimulated but not resting macrophages elicited HIF-1alpha 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-alpha (TNF-alpha )-neutralizing antibodies, we identified NO and TNF-alpha as signaling molecules. Working in concert, NO and TNF-alpha 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-alpha 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-alpha , derived from activated macrophages, provoke HIF-1alpha stabilization in LLC-PK1 cells under normoxic conditions, which underscores HIF-1alpha stabilization due to intercellular regulation.

nitric oxide; tumor necrosis factor-alpha ; hypoxia-inducible factor-1; intercellular signaling; phosphatidylinositol 3-kinase; cytokine; Akt


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha and the aryl hydrocarbon nuclear translocator that is also known as HIF-1beta (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-1alpha 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-1alpha (17, 37).

The availability of HIF-1 is predominantly determined by stability regulation of HIF-1alpha via proline hydroxylation (19, 20, 39). HIF-1alpha 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-1alpha 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-1alpha positions 564 and/or 402 to hydroxylate HIF-1alpha (19, 20). Proline hydroxylation appears to be necessary and sufficient for the binding of pVHL to HIF-1alpha with concomitant degradation of HIF-1alpha 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-1alpha 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-1alpha 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-1alpha 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-1alpha stability (34, 35). Reports on cytokines in HIF-1alpha stability regulation have indicated that interleukin-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ) are positively involved (16, 32, 40). The term stabilization refers to a situation whereby the HIF-1alpha 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-1alpha 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-1alpha stability regulation. We provide evidence by using a coculture setup whereby activated macrophages release NO and TNF-alpha as soluble messengers that provoke an HIF-1alpha 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha 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 pSRalpha -Delta p85 and pSRalpha -WTp85 were kindly provided by Dr. W. Ogawa (School of Medicine, Kobe University, Kobe, Japan; see Ref. 15). Plasmids pCMV5 and pCMV5.-m/p-PKBalpha 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-gamma (INF-gamma , 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-alpha 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.

In some experiments, cells were cocultured by using Transwell inserts with a 1-µm porous membrane to separate the cells. For these experiments, 1 × 107 RAW 264.7 cells were stimulated with LPS (1 µg/ml) and INF-gamma (100 U/ml) for 18 h in 10-cm dishes. Cells were washed with PBS, scraped from the dishes, collected by centrifugation, and replated [in the presence or absence of 1 mM NG-nitro-L-arginine methyl ester (L-NAME)] in the bottom of six-well plates. At the same time, 4 × 105 LLC-PK1 cells seeded on coculture inserts were added, and cells were cocultured for 12 h before performance of HIF-1alpha detection in LLC-PK1 cells.

Western blot analysis. HIF-1alpha , 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-1alpha 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-alpha determinations. As previously described (33), detection of TNF-alpha in culture supernatants was performed on flat-bottomed, high-binding, polystyrene microtiter plates (Nunc, Roskilde, Denmark) using specific monoclonal antibody pairs (anti-mouse TNF-alpha and biotinylated anti-mouse TNF-alpha ) 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-1alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Activated macrophages provoke HIF-1alpha accumulation in LLC-PK1 cells. During inflammation, macrophages are activated and proinflammatory mediators are released. To study HIF-1alpha 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-1alpha 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-gamma . First, we ensured that no HIF-1alpha 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-1alpha response as well as nitrite formation. Because RAW 264.7 cells did not stabilize HIF-1alpha in response to LPS/IFN-gamma addition, it must be concluded that the HIF-1alpha 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-1alpha 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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Fig. 1.   Activated RAW 264.7 macrophages provoke hypoxia-inducible factor-1alpha (HIF-1alpha ) accumulation in LLC-PK1 cells via the release of nitric oxide (NO). RAW 264.7 macrophages were stimulated with vehicle (lane I, control macrophages), the combination of 1 µg/ml lipopolysaccharide (LPS) and 100 U/ml interferon-gamma (IFN-gamma ; lane II, activated macrophages) for 18 h, or LPS with IFN-gamma for 18 h with the addition of 1 mM NG-nitro-L-arginine methyl ester (L-NAME) for 8 h (lane III). In the coculture setup, LLC-PK1 cells were cocultured with control macrophages (lane IV) or activated macrophages (lane V) for an 8-h period. Cocultures were carried out with the addition of 1 mM L-NAME (lane VI) or 100 µM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; lane VII). For control reasons, macrophages were stimulated with 25 µM cisplatin for 18 h and then cocultured with LLC-PK1 cells for 8 h (lane VIII). HIF-1alpha protein was detected by Western blot analysis as outlined (see Western blot analysis). Nitrite formation during the time of coculture was measured via the Griess method. Experiments were performed at least three times, and representative data are shown.

In addition to L-NAME, we used 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) to scavenge NO in the medium and thereby downregulate HIF-1alpha stabilization in the coculture setup. Because it is known that stimulation of RAW 264.7 macrophages for 18 h with LPS/IFN-gamma provokes apoptotic cell death, it was necessary to rule out that the HIF-1alpha stabilization was elicited by apoptotic cells in general. Therefore, macrophages were exposed for 18 h to 25 µM cisplatin, which was followed by an 8-h coculture with LLC-PK1 cells (Fig. 1). This maneuver caused no HIF-1alpha response in LLC-PK1 cells. We conclude that activated macrophages provoke HIF-1alpha stabilization in tubular LLC-PK1 cells with the notion that signal transmission is attenuated by the blocking or scavenging of NO. For statistical evaluation, we performed densitometry. QLs of the HIF-1alpha bands under control conditions gave values ranging from 0.2 × 103 to 1.01 × 105, whereas values from 1.8 × 106 to 3.5 × 106 were noticed for the stimulated situation. Expressing values for HIF-1alpha as an increase compared with controls must be approached with caution considering that HIF-1alpha basically is absent under control conditions. Having these drawbacks in mind, we measured at least a 20 ± 4-fold increase in HIF-1alpha expression (mean ± SE, n >=  5) compared with controls.

To avoid direct cell-to-cell contact, we used Transwell inserts that contained a 1-µm porous membrane. LLC-PK1 cells (4 × 105) were seeded on the porous membrane, whereas LPS/INF-gamma -activated RAW 264.7 cells (1 × 107) were plated at the bottom of a six-well plate. After a 12-h coculture period, LLC-PK1 cells were recovered, and HIF-1alpha accumulation was analyzed (Fig. 2). With resting macrophages, we noticed a very minor HIF-1alpha signal in LLC-PK1 cells (lane I), whereas LPS/INF-gamma -activated macrophages elicited a robust, roughly 18-fold increased signal (lane II).


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Fig. 2.   Activated RAW 264.7 macrophages provoke HIF-1alpha accumulation in LLC-PK1 cells using a Transwell setup. A: RAW 264.7 macrophages (placed in the bottom of Transwell plates) were cocultured with LLC-PK1 cells (placed in the inserts) for 12 h, and HIF-1alpha was detected by Western blot analysis in LLC-PK1 cells. Unstimulated macrophages (lane I), LPS/IFN-gamma -activated macrophages (lane II), or activated macrophages in the presence of L-NAME (lane III) were used. B: densitometric analyses of HIF-1alpha signals (means ± SE, n = 3) in proportion to the actin signal [relative quantum level (QL)] were performed as described (see Densitometric quantification and statistical analysis).

Attenuating NO formation with L-NAME (lane III) reduced HIF-1alpha accumulation in LLC-PK1 cells drastically and left only a roughly twofold increase over controls. We conclude that directly coculturing macrophages with LLC-PK1 cells or coculturing cells separated by a porous membrane allowed the generation of signals in macrophages that evoked HIF-1alpha accumulation in LLC-PK1 cells.

We then investigated signaling pathways that are required to stabilize HIF-1alpha in LLC-PK1 cells when cocultured with activated macrophages. In accordance with the concept that PI3-K and Akt might be involved, we transfected LLC-PK1 cells with a dominant-negative p85 form (Delta p85) of PI3-K, which lacks amino acids 479-513, or used wortmannin, which is an established inhibitor of PI3-K (Fig. 3). As is similarly shown in Figs. 1 and 2, HIF-1alpha was stabilized in LLC-PK1 cells when cocultured with activated but not resting macrophages (at least a 20 ± 4-fold increase in HIF-1alpha expression; mean ± SE, n >=  3 compared with controls).


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Fig. 3.   Inactivation of phosphatidylinositol 3-kinase (PI3-K) signaling attenuated HIF-1alpha signals in LLC-PK1 cells. LLC-PK1 cells (4 × 105) were transfected with 3 µg of pSRalpha -Delta p85 (Delta p85, lane 3) or the corresponding empty vector plasmid or were prestimulated for 30 min with 100 nM wortmannin (WT, lane 4). LLC-PK1 cells were then cocultured for 8 h with nonactivated (C, control) or activated (L/I, 1 µg/ml LPS and 100 U/ml IFN-gamma for 18 h) macrophages. Accumulation of HIF-1alpha (top assay) and p85 (bottom assay) were determined by Western blot analysis. Each experiment was performed at least three times, and representative data are shown. Densitometric analysis is given as the QL of corresponding HIF-1alpha bands.

When the PI3-K pathway in LLC-PK1 cells was turned off by transfection of Delta p85, stabilization of HIF-1alpha was eliminated. The HIF-1alpha signal was reduced by 95 ± 3% (mean ± SE, n>= 3). We obtained similar results when wortmannin was preincubated for 30 min with LLC-PK1 cells before the addition of activated macrophages. As a further indication of the involvement of the PI3-K/Akt pathway, we determined activation of Akt by using a phospho-specific antibody that recognizes only phosphorylated serine 473 (Fig. 4A). A coculture of LLC-PK1 and RAW cells revealed no phosphorylation and thus no activation of Akt.


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Fig. 4.   Activation of Akt and attenuation of HIF-1alpha signals due to inactive Akt. LLC-PK1 cells were cocultured for 4-8 h with nonstimulated (C, control) or activated (L/I, 1 µg/ml LPS and 100 U/ml IFN-gamma for 18 h) RAW 264.7 macrophages. A: activation of Akt was determined by Western blot analysis using anti-phospho serine 473 antibodies. B: LLC-PK1 cells (4 × 105) were transfected with 3 µg of pCMV5.-m/p-PKBalpha K179 (PKBK179) or the corresponding empty vector/plasmid before coculture. Accumulation of HIF-1alpha and expression of Akt were determined by Western blot analysis. Each experiment was performed at least three times, and representative data are shown.

When activated macrophages were added to LLC-PK1 cells, we determined phosphorylation of Akt after 4 h with a stronger signal after 6 h, which further increased at 8 h. To provide evidence that activation of Akt indeed is involved, we eliminated Akt in LLC-PK1 cells by transfection of a dominant-negative Akt protein (Fig. 4B). Dysfunctional Akt was achieved by mutating the ATP-binding site lysine 179 to alanine (3). In addition, a consensus sequence for both myristoylation and palmitylation (m/p) was hooked to the construct as activated Akt is recruited to the membrane via its pleckstrin homology (PH) domain. M/p has been shown to be sufficient to localize a number of cytosolic proteins to the plasma membrane (44). Therefore, Akt activation is blocked but its translocation is not (3). Control experiments with the empty pCMV5 vector showed no interference with HIF-1alpha accumulation (data not shown).

However, overexpression of PKBalpha K179A suppressed HIF-1alpha stabilization in LLC-PK1 detector cells. The signal was reduced by 94 ± 4% (mean ± SE, n >=  3). Western blot analysis confirmed overexpression of PKBalpha K179A compared with constitutive expression of endogenous Akt.

Macrophages use multiple signals to stabilize HIF-1alpha in LLC-PK1 cells. In searching for HIF-1alpha -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-1alpha stabilization.


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Fig. 5.   Accumulation of HIF-1alpha by soluble messengers. RAW 264.7 macrophages were activated for 18 h with 1 µg/ml LPS and 100 U/ml IFN-gamma (L/I) or were left unstimulated (C, control). Thereafter, macrophages were cocultured with LLC-PK1 cells (cells) for 8 h or only the supernatant of activated macrophages was added to LLC-PK1 cells (medium). A: HIF-1alpha was detected by Western blot analysis. B: in either case (Cells/Ab and Medium/Ab), TNF-alpha was neutralized by the addition of a TNF-alpha antibody (Ab). HIF-1alpha was detected by Western blot analysis. Experiments were performed at least three times, and representative data are shown.

When the cell supernatant from activated macrophages was transferred to LLC-PK1 cells for a subsequent 8-h incubation period, the HIF-1alpha response was markedly reduced but still visible. As expected, the supernatant from nonactivated macrophages was unable to provoke an HIF-1alpha response.

Considering the importance of TNF-alpha and NO as proinflammatory markers of activated macrophages, and taking into consideration that both agonists may provoke an HIF-1alpha response, we determined the amounts of NO and TNF-alpha in the supernatant of RAW 264.7 cells. Under resting conditions, macrophages produced 1.1 ± 1 pg/ml TNF-alpha and ~1 µM nitrite during an 18-h sampling period. When stimulated with LPS/IFN-gamma for 18 h, TNF-alpha amounted to 547 ± 55 pg/ml and nitrite values rose to 36 ± 10 µM.

In the following set of experiments, we delineated the role of TNF-alpha when released from activated macrophages in stabilizing HIF-1alpha in LLC-PK1 detector cells (Fig. 5B). Therefore, we used TNF-alpha -neutralizing antibodies in the macrophage/LLC-PK1 coculture setup. Macrophages were activated with LPS/IFN-gamma for 18 h before direct coculture with detector cells, which continued for 8 h. Under these experimental conditions, HIF-1alpha was stabilized in LLC-PK1 cells. Interestingly, addition of TNF-alpha -neutralizing antibodies during the time of the coculture largely attenuated HIF-1alpha stabilization. Inhibition amounted to 92 ± 5% (mean ± SE, n >=  3). A similar response was observed when direct cell-to-cell contacts between donor and detector cells were avoided by adding the cell supernatant of activated macrophages to LLC-PK1 cells. The supernatant of LPS/IFN-gamma -stimulated macrophages provoked an HIF-1alpha signal in LLC-PK1 detector cells that again was sensitive to the addition of TNF-alpha -neutralizing antibodies. TNF-alpha in the supernatant of control vs. LPS/IFN-gamma -activated RAW 264.7 cells amounted to 1 ± 1 pg/ml in unstimulated cells and increased to 64 ± 10 pg/ml during an 8-h sampling period that started 18 h after stimulation with LPS/IFN-gamma . Addition of TNF-alpha -neutralizing antibodies during the 8-h sampling period reduced the level of free TNF-alpha to 2 ± 4 pg/ml, whereas nitrite was not affected (data not shown). Based on these results, TNF-alpha and NO need consideration as intercellular messengers that add to the accumulation of HIF-1alpha .

Having established that TNF-alpha is a major cytokine in our experimental system, we went on to demonstrate HIF-1alpha accumulation in LLC-PK1 cells under the impact of recombinant TNF-alpha (Fig. 6A). The time dependency of TNF-alpha (at a concentration of 500 ng/ml) showed a slow response. HIF-1alpha accumulated not before 8 h but remained stabilized up to 16 h.


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Fig. 6.   HIF-1alpha accumulation under the impact of TNF-alpha and diethylenetriamine-nitric oxide (DETA-NO). A: LLC-PK1 cells were stimulated with 500 ng/ml TNF-alpha and 500 µM DETA-NO for times indicated or were left untreated (C, control). B: LLC-PK1 cells were incubated with 500 µM DETA-NO and 500 ng/ml TNF-alpha for 6 h either alone or in combination. Accumulation of HIF-1alpha was detected by Western blot analysis. Experiments were performed at least three times, and representative data are shown.

This response was not altered at elevated concentrations of TNF-alpha (1.5 µg/ml; data not shown). In comparison, NO derived from 500 µM DETA-NO provoked a faster response. HIF-1alpha accumulated within 2 h, reached a maximum after 4-6 h, and levels declined thereafter. Evidently, NO and TNF-alpha share the ability to stabilize HIF-alpha but must be regarded as fast vs. slow HIF-alpha inducers.

In the last set of experiments, we determined the ability of NO and TNF-alpha to act in concert (Fig. 6B). For these experiments, we chose a 6-h time point. This allows NO to achieve a maximal effect, whereas TNF-alpha still appears inactive. DETA-NO caused a moderated HIF-1alpha signal at 6 h. TNF-alpha alone induced no HIF-1alpha signal at these early time points, but in combination with NO, enhanced HIF-1alpha protein accumulation ~2.3 ± 0.5-fold (mean ± SE, n = 3). We conclude that both NO and TNF-alpha are effective regulators of HIF-1alpha stabilization.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

By coculturing macrophages and tubular LLC-PK1 cells, we established that HIF-1alpha is subjected to stability regulation by soluble intercellular messengers. NO and TNF-alpha released from activated but not resting macrophages stabilized HIF-1alpha in LLC-PK1 detector cells. Activation of intracellular signaling pathways as a result of NO and TNF-alpha 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-1alpha 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-alpha , growth factors, or hormones (1, 6, 8, 13, 30, 35, 42). Although diverse factors stimulate HIF-1alpha accumulation and activation in a variety of cell types, we still lack detailed knowledge about how intercellular signaling mechanisms converge in HIF-1alpha stability regulation and whether individual agonists combine effects. Most likely, multiple signaling pathways are capable of modulating HIF-1alpha 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-1alpha 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-1alpha . In our study, we proceeded to demonstrate accumulation of HIF-1alpha due to intercellular signaling pathways. This opens the possibilities of autocrine- and paracrine-stability regulation of HIF-1alpha . Thus the HIF-1alpha -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-1alpha accumulation in LLC-PK1 detector cells. NO and TNF-alpha 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-alpha -neutralizing antibodies. Blocking and/or eliminating either NO or TNF-alpha abrogated signal transmission to a large extent. We must conclude that under coculture conditions, only the combined biological activity of NO and TNF-alpha achieves maximal HIF-1alpha accumulation. This is somewhat in analogy to the study of Hellwig-Bürgel et al. (16), who found that cytokines such as IL-1beta or TNF-alpha 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-1alpha protein accumulation when treating LLC-PK1 cells with a combination of DETA-NO and TNF-alpha , although NO donors as well as TNF-alpha are known to be self-sufficient in eliciting an HIF-1alpha response (1, 12, 13, 22, 31, 32). When elicited by activated macrophages, HIF-1alpha stabilization in detector cells makes use of established and known signaling pathways. However, blocking either the NO or the TNF-alpha portion of the signal abrogated HIF-1alpha 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-1alpha 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-1alpha response.

In the particular case of TNF-alpha 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-alpha 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-1alpha stabilization in response to TNF-alpha . Despite the contradiction on the role of PI3-K in HIF-1alpha stabilization during hypoxia, NO and TNF-alpha 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-1alpha (10, 43). Future work needs to address how a PI3-K signal manages to attenuate HIF-1alpha degradation and whether activation of nuclear factor-kappa B or activator protein-1, two well-established downstream signaling pathways of TNF-alpha , are involved.

Considering NO and TNF-alpha as important intercellular messengers that provoke an HIF-1alpha response, it may be justified to put their formation and action into a pathophysiological, i.e., inflammatory and/or tumorgenic, context. TNF-alpha is a potent proinflammatory cytokine, and NO can be considered a marker of inflammatory host defense. Formation of TNF-alpha and NO is essential for cell proliferation, differentiation, and apoptosis, and thus HIF-1alpha 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-1alpha 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-1alpha by intercellular messengers makes perfect sense.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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