Bernhard Brüne

HIF-1 and p53: communication of transcription factors under hypoxia.

Oxygen sensing and reactivity to changes in the concentration of oxygen is a fundamental property of cell physiology. The lack of O2 (hypoxia) is transmitted into many adaptive responses, a process that is largely controlled by a transcription factor known as hypoxia inducible factor‐1 (HIF‐1). More recent reports suggest that besides its traditional regulation via proteasomal degradation other signaling pathways contribute to stability regulation of the HIF‐1α subunit and/or HIF‐1 transactivation. These regulatory circuits allow for the integration of HIF‐1 into scenarios of cell‐survival vs. cell‐death with the rule of the thumb that short‐term mild hypoxia maintains cell viability while prolonged and severe hypoxia provokes cell demise. Cell death pathways are associated with stabilization of the tumor suppressor p53, a response also seen under hypoxic conditions. Here we summarize recent information on accumulation of HIF‐1α and p53 under hypoxia and provide a model to explain the communication between HIF‐1 and p53 under (patho)physiological conditions.

HIF-1α protein as a target for S-nitrosation

Hypoxia‐inducible factor‐1α (HIF‐1α) is a master regulator to sense decreased oxygen partial pressure. HIF‐1α stability regulation initiates a complex biological response that allows cells to act appropriately to meet patho‐physiological situations of decreased oxygen availability. Recently, nitric oxide emerged as a messenger with the ability to stabilize HIF‐1α and to transactivate HIF‐1 under normoxia. Considering that reactive nitrogen species are recognized for post‐translation protein modifications, among others S‐nitrosation, we asked whether HIF‐1α is a target for S‐nitrosation. In vitro NO+ donating NO donors such as GSNO and SNAP provoked massive S‐nitrosation of purified HIF‐1α. All 15 free thiol groups found in human HIF‐1α are subjected to S‐nitrosation. Thiol modification is not shared by spermine‐NONOate, a NO radical donating compound. However, spermine‐NONOate in the presence of O2 −, generated by xanthine/xanthine oxidase, regained S‐nitrosation, most likely via formation of a N2O3‐like species. In vitro, S‐nitrosation of HIF‐1α was attenuated by the addition of GSH or ascorbate. In RCC4 and HEK293 cells GSNO or SNAP reproduced S‐nitrosation of HIF‐1α, however with a significantly reduced potency that amounted to modification of three to four thiols, only. Importantly, endogenous formation of NO in RCC4 cells via inducible NO synthase elicited S‐nitrosation of HIF‐1α that was sensitive to inhibition of inducible NO synthase activity with N‐monomethyl‐L‐arginine. NO‐stabilized HIF‐1α was susceptible to the addition of N‐acetyl‐cysteine that destabilized HIF‐1α in close correlation to the disappearance of S‐nitrosated HIF‐1α. In conclusion, HIF‐1α is a target for S‐nitrosation by exogenously and endogenously produced NO.

p300 relieves p53-evoked transcriptional repression of hypoxia-inducible factor-1 (HIF-1).

HIF-1 (hypoxia-inducible factor-1), a heterodimeric transcription factor comprising HIF-1alpha and HIF-1beta subunits, serves as a key regulator of metabolic adaptation to hypoxia. HIF-1 activity largely increases during hypoxia by attenuating pVHL (von Hippel-Lindau protein)-dependent ubiquitination and subsequent 26 S-proteasomal degradation of HIF-1alpha. Besides HIF-1, the transcription factor and tumour suppressor p53 accumulates and is activated under conditions of prolonged/severe hypoxia. Recently, the interaction between p53 and HIF-1alpha was reported to evoke HIF-1alpha degradation. Destruction of HIF-1alpha by p53 was corroborated in the present study by using pVHL-deficient RCC4 (renal carcinoma) cells, supporting the notion of a pVHL-independent degradation process. In addition, low p53 expression repressed HIF-1 transactivation without affecting HIF-1alpha protein amount. Establishing that p53-evoked inhibition of HIF-1 reporter activity was relieved upon co-transfection of p300 suggested competition between p53 and HIF-1 for limiting amounts of the shared co-activator p300. This assumption was confirmed by showing competitive binding of in vitro transcription/translation-generated p53 and HIF-1alpha to the CH1 domain of p300 in vitro. We conclude that low p53 expression attenuates HIF-1 transactivation by competing for p300, whereas high p53 expression destroys the HIF-1alpha protein and thereby eliminates HIF-1 reporter activity. Thus once p53 becomes activated under conditions of severe hypoxia/anoxia, it contributes to terminating HIF-1 responses.

Nitric oxide donors inhibit formation of the Apaf-1/caspase-9 apoptosome and activation of caspases.

Caspases are critical for the initiation and execution of apoptosis. Nitric oxide (NO) or derived species can prevent programmed cell death in several cell types, reportedly through S-nitrosation and inactivation of active caspases. Although we find that S-nitrosation of caspases can occur in vitro, our study questions whether this post-translational modification is solely responsible for NO-mediated inhibition of apoptosis. Indeed, using Jurkat cells as a model system, we demonstrate that NO donors block Fas- and etoposide-induced caspase activation and apoptosis (downstream of mitochondrial membrane depolarization) and cytochrome c release. However, caspase activity was not restored by the strong reducing agent dithiothreitol, as predicted for S-nitrosation reactions, thereby excluding active-site-thiol modification of caspases as the only anti-apoptotic mechanism of NO donors in cells. Rather, we observed that processing of procaspases-9, -3 and -8 was decreased due to ineffective formation of the Apaf-1/caspase-9 apoptosome. Gel-filtration and in vitro binding assays indicated that NO donors inhibit correct assembly of Apaf-1 into an active approx. 700 kDa apoptosome complex, and markedly attenuate caspase-recruitment domain (CARD)-CARD interactions between Apaf-1 and procaspase-9. Therefore we suggest that NO or a metabolite acts directly at the level of the apoptosome and inhibits the sequential activation of caspases-9, -3 and -8, which are required for both stress- and receptor-induced death in cells that use the mitochondrial subroute of cell demise.

Activation of Peroxisome Proliferator-Activated Receptor γ by Nitric Oxide in Monocytes/Macrophages Down-Regulates p47phox and Attenuates the Respiratory Burst

NO appears as an important determinant in auto and paracrine macrophage function. We hypothesized that NO switches monocyte/macrophage function from a pro- to an anti-inflammatory phenotype by activating anti-inflammatory properties of the peroxisome proliferator-activated receptor (PPAR)γ. NO-releasing compounds (100 μM S-nitrosoglutathione or 50 μM spermine-NONOate) as well as inducible NO synthase induction provoked activation of PPARγ. This was proven by EMSAs, with the notion that supershift analysis pointed to the involvement of PPARγ. PCR analysis ruled out induction of PPARγ mRNA as a result of NO supplementation. Reporter assays, with a construct containing a triple PPAR response element in front of a thymidine kinase minimal promoter driving the luciferase gene, were positive in response to NO delivery. DNA binding capacity as well as the transactivating capability of PPARγ were attenuated by addition of the antioxidant N-acetyl-cysteine or in the presence of the NO scavenger 2-phenyl-4,4,5,6-tetramethyl-imidazoline-1-oxyl 3-oxide. Having established that NO but not lipophilic cyclic GMP analogs activated PPARγ, we verified potential anti-inflammatory consequences. The oxidative burst of macrophages, evoked by phorbol ester, was attenuated in association with NO-elicited PPARγ activation. A cause-effect relationship was demonstrated when PPAR response element decoy oligonucleotides, supplied in front of NO delivery, allowed to regain an oxidative response. PPARγ-mediated down-regulation of p47 phagocyte oxidase, a component of the NAD(P)H oxidase system, was identified as one molecular mechanism causing inhibition of superoxide radical formation. We conclude that NO participates in controlling the pro- vs anti-inflammatory phenotype of macrophages by modulating PPARγ.

Dualism of Oxidized Lipoproteins in Provoking and Attenuating the Oxidative Burst in Macrophages: Role of Peroxisome Proliferator-Activated Receptor-γ

Activation and deactivation of macrophages are of considerable importance during the development of various disease states, atherosclerosis among others. Macrophage activation is achieved by oxidized lipoproteins (oxLDL) and is determined by oxygen radical (ROS) formation. The oxidative burst was measured by flow cytometry and quantitated by oxidation of the redox-sensitive dye dichlorodihydrofluorescein diacetate. Short-time stimulation dose-dependently elicited ROS formation. Diphenylene iodonium prevented ROS formation, thus pointing to the involvement of a NAD(P)H oxidase in producing reduced oxygen species. In contrast, preincubation of macrophages with oxLDL for 16 h showed an attenuated oxidative burst upon a second contact with oxLDL. Taking into account that oxLDL is an established peroxisome proliferator-activated receptor-γ (PPARγ) agonist and considering the anti-inflammatory properties of PPARγ, we went on and showed that a PPARγ agonist such as ciglitazone attenuated ROS formation. Along that line, major lipid peroxidation products of oxLDL, such as 9- and 13-hydroxyoctadecadienoic acid, shared that performance. Supporting evidence that PPARγ activation accounted for reduced ROS generation came from studies in which proliferator-activated receptor response element decoy oligonucleotides, but not a mutated oligonucleotide, supplied in front of oxLDL delivery regained a complete oxidative burst upon cell activation. We conclude that oxLDL not only elicits an oxidative burst upon first contact, but also promotes desensitization of macrophages via activation of PPARγ. Desensitization of macrophages may have important consequences for the behavior of macrophages/foam cells in atherosclerotic lesions.

Nitric oxide induces phosphorylation of p53 and impairs nuclear export

The tumor suppressor p53 accumulates under diverse stress conditions and affects cell cycle progression and/or apoptosis. This has been exemplified for endogenously produced or exogenously supplied nitric oxide (NO) and thus accounts at least in part for pathophysiological signaling of that bioactive molecule, although detailed mechanisms remain to be elucidated. By using luciferase reporter assays, we show that NO stabilized a transcriptionally active p53 protein. Considering that p53 is targeted by murine double minute (Mdm2) for ubiquitination and subsequent proteasomal degradation and knowing that this interaction is impaired by, for example, UV-treatment with concomitant stabilization of p53 we questioned the p53/Mdm2 interaction in the presence of NO. Although p53 became phosphorylated at serine 15 under the impact of NO, coimmunoprecipitation with Mdm2 and ubiquitination remained intact, thus excluding any interference of NO with this pathway. The importance of N-terminal p53 phosphorylation was verified with p53 mutants where the first six serine residues have been converted to alanine, and which do not accumulate in response to NO. Regulation of p53 stability can be also achieved by affecting nuclear–cytoplasmic shuttling and it was presented that leptomycin B, an inhibitor of nuclear export, caused p53 accumulation. Cell fractionation and immunofluorescence staining following NO-treatment revealed predominant nuclear accumulation of p53 in close association with serine 15-phosphorylation, which suggests impaired nuclear–cytoplasmic shuttling. This was verified by heterokaryon analysis. We conclude that attenuated nuclear export contributes to stabilization and activation of p53 under the influence of NO.

Nitric oxide prevents oxidised LDL-induced p53 accumulation, cytochrome c translocation, and apoptosis in macrophages via guanylate cyclase stimulation

BACKGROUND Oxidatively modified low density lipoprotein (OxLDL) induces apoptosis in vascular cells including macrophages, while NO exerts antiapoptotic effects. Here we studied the impact of nitric oxide (NO) on OxLDL-induced cytochrome c release, apoptosis, and expression of the proapoptotic p53 in macrophages. METHODS Human LDL was oxidised by Cu(2+), and monocytes were prepared from human buffy coats. Differentiation to macrophages was achieved by culturing cells in the presence of human serum and was followed by detecting monocyte chemoattractant protein 1 (MCP-1) expression (RT-PCR). Cytochrome c release and p53 expression of macrophages were detected by immunoblotting, and apoptosis by visualisation of nuclear condensation. RESULTS OxLDL dose-dependently (50-200 microg/ml) induced cytochrome c release that was prevented by preincubation with the NO-donor S-nitrosoglutathione (GSNO) (100 microM) or with the cGMP analogue 8-br-cGMP (100 microM) for 15 h. In cells co-treated with GSNO and the soluble guanylate cyclase (sGC) inhibitor oxadialoquinoxalione (ODQ, 10 microM, 15 h), OxLDL-evoked cytochrome c release remained effective, indicating that NO acted via sGC-dependent cGMP formation. Parallel incubation of macrophages with 8-br-cGMP (100 microM) and ODQ (10 microM) for 15 h left the protective effect of 8-br-cGMP unaltered. Short pre-incubation (30 min) with GSNO or 8-br-cGMP was ineffective in preventing OxLDL-elicited cytochrome c release. Initiation of cytochrome c release in macrophages was paralleled by a dose-dependent accumulation of the proapoptotic factor p53, and by enhanced rate of nuclear condensation. Stabilisation of p53 was prevented by preincubation with the NO-donor GSNO or 8-br-cGMP, thus implying a downmodulatory effect of cGMP on pathways that upregulate the tumor suppressor p53. CONCLUSIONS OxLDL induces cytochrome c release and apoptosis in human macrophages in close association with p53 accumulation. NO attenuates OxLDL-induced cytochrome c release and p53 accumulation via activation of sGC and cGMP formation. These effects may be of particular importance in arterial tissue with reduced NO activity.

Heat-shock protein 70 attenuates nitric oxide-induced apoptosis in RAW macrophages by preventing cytochrome c release

Heat-shock protein (Hsp) 70 is an inhibitor of apoptosis and has been shown to protect against nitric oxide-mediated toxicity. To gain mechanistic insights into the actions of Hsp70, we stably transfected RAW 264.7 mouse macrophages with the human Hsp70 gene and investigated critical steps in the progression towards cell demise. Incubation of control and Hsp70-transfected macrophages with S-nitrosoglutathione induced accumulation of the tumour suppressor p53, expression of p21(WAF1/CIP1) (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1) and G(1) cell-cycle arrest. However, cytochrome c translocation to the cytosol and activation of caspase 9 and caspase 3 were markedly reduced in Hsp70-overexpressing cells. In addition, changes in nuclear morphology, as determined by Hoechst staining, and the appearance of cells in the sub-G(1) phase were diminished in Hsp70-overexpressing cells compared with controls. We conclude that, in macrophages, Hsp70 interferes with cytochrome c release from mitochondria and, thereby, prevents nitric oxide-induced apoptosis, but leaves p53 accumulation and interference in the cell cycle intact.

Activation-induced PPARγ expression sensitizes primary human T cells toward apoptosis

Phytohemagglutinin (PHA) elicited expression of peroxisome proliferator‐activated receptor γ (PPARγ) in primary human T cells via the PPARγ3 promoter, as shown by reverse transcription‐polymerase chain reaction. Electrophoretic mobility shift assay demonstrated no correlation between PPARγ expression and its activation. However, addition of specific PPARγ agonists such as ciglitazone or 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) for 1 h following PHA pretreatment provoked PPARγ activation verified by supershift analysis. Taking the proapoptotic properties of PPARγ into consideration, we analyzed induction of apoptosis in activated T cells in response to PPARγ agonists. Cells exposed to PPARγ agonists alone revealed minor cell death compared with controls, whereas treatment with 15d‐PGJ2 or ciglitazone for 4 h subsequent to PHA stimulation significantly increased cell demise, which was attenuated by the pan‐caspase inhibitor zVAD, pointing to apoptosis as the underlying mechanism. These data may be relevant for pathophysiological conditions accompanied with lymphopenia of T cells under conditions such as sepsis.