Ionizing radiation affects 26s proteasome function and associated molecular responses, even at low doses

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Abstract

Background and purpose: Ionizing radiation is known to activate certain signal transduction pathways, the regulation of which could involve post-transcriptional as well as transcriptional mechanisms. One of the most important post-transcriptional pathways in eukaryotic cells is the ATP- and ubiquitin-dependent degradation of proteins by the 26s proteasome. This process controls initiation of many cellular stress responses, as well as inflammatory responses under control of the transcription factor NF-κB. The literature on the relationship between radiation and inflammation seems somewhat paradoxical. At high doses, radiation is generally pro-inflammatory. On the other hand, low dose radiation has a long history of use in the treatment of inflammatory disease. This suggests the involvement of multiple mechanisms that may operate differentially at different dose levels.

Materials and methods: In this paper, the ability of different doses of ionizing radiation to directly affect 26s proteasome activity was tested in ECV 304 cells. Proteasome activity, IκBα protein levels, and NF-κB activation were monitored.

Results: Inhibition of chymotrypsin-like 20s and 26s proteasome activity was observed immediately after low- and high-dose irradiation either of cells or purified proteasomes. The inhibitory effect was independent of the availability of the known endogenous proteasome inhibitor heat shock protein 90 (hsp90). Levels of IκBα, a physiological 26s proteasome substrate, were increased only at low doses (0.25 Gy) and unaltered at higher doses whereas only the highest doses (8 and 20 Gy) activated NF-κB.

Conclusions: We conclude that the proteasome is a direct target of ionizing radiation and suggest that inhibition of proteasome function provides a molecular framework within which low dose anti-inflammatory effects of radiation, and radiation-induced molecular responses in general, should be considered.

Introduction

In recent years considerable interest has been shown in the mechanisms by which ionizing radiation induces early molecular responses and how these might influence subsequent radiation-related events [8], [16]. Cellular gene expression can be re-orchestrated by radiation within minutes to hours. The most frequently reported immediate early responses include up-regulation of expression of the transcription factors JUN, FOS, and NF-κB, [2], [4], [14], [15], [20], [28], [43], [44], [47], [50] and of molecules implicated in recognition and repair of damaged DNA, in responses to oxidative stress, and in cell cycle arrest and death [16]. These immediate early responses co-ordinately link radiation damage to pathways that promote wound healing and tissue remodeling [8].

Gene expression can be modified by post-transcriptional as well as transcriptional mechanisms. The former are generally the more rapid. NF-κB, which is the prime player linking radiation and other signals to inflammatory responses, is a good example. NF-κB is a hetero- or homodimer of the subunits p50, p52, p65/RelA, c-Rel, and Rel-B. It is sequestered preformed in the cytosol by inhibitor molecules of the IκB family (IκBα, IκBβ, IκBγ, Bcl-3, p100, and p105). Activation of this pathway is normally achieved by phosphorylation of one of the most important inhibitors, IκBα, at two serine-sites (Ser-32 and Ser-36) by IκB kinases. This marks IκBα for polyubiquitination and subsequent degradation by the 26S proteasome. Degradation of IκBα frees NF-κB for translocation to the nucleus and activation of its target genetic programs (reviewed in [3]).

NF-κB activation in response to pro-inflammatory agents such as TNFα is a redox-sensitive process [42]. It is therefore not surprising that ionizing radiation can activate NF-κB [31] through the classical ubiquitin/proteasome-dependent pathway [7], [26], although in astrocytes and human brain tumor cells, phosphorylation of IκBα at tyrosine residues without its subsequent degeneration has been suggested as an alternate pathway [39]. In lymphocytes, NF-κB activation that is non-linear with dose has been reported [31], [26], and this is the case for certain other radiation-induced molecular responses [43]. It seems likely that this non-linearity is the result of multiple mechanisms that operate differentially with dose.

Because ubiquitination and degradation of negative regulatory molecules through the proteasome is often a first critical step for activation of many molecular pathways, we have investigated the possibility the proteasome itself is a redox-sensitive target for irradiation. The 26s proteasome is responsible for the controlled ATP-and ubiquitin-dependent degradation of all short-lived [9] and 70–90% of all long-lived proteins [9], [25], including key molecules in signal transduction, cell cycle control, and immune response [41]. Recently, it has become clear that this activity can be intrinsically regulated. Here, we provide evidence that the 26s proteasome complex is a direct target of ionizing radiation and that this mechanism operates functionally to inhibit activation of NF-κB at low radiation doses. It is of interest that although high dose radiation is generally pro-inflammatory [30], the history of radiation therapy for benign diseases in particular is replete with examples where ionizing radiation was used to terminate preexisting inflammatory conditions [45]. The molecular basis for this paradox is unexplained, although low radiation doses have been shown to inhibit nitric oxide production by macrophages, while high doses result in super-stimulation [18]. The relative contributions of post-transcriptional and transcriptional control mechanisms could help to explain some of the immediate early molecular effects of ionizing radiation at different dose levels.

Section snippets

Cell culture

Cultures of ECV 304 human bladder carcinoma cells (ATCC) and RAW 264.7 murine macrophages (a generous gift of Dr G. Hildebrandt, Department of Radiation Oncology, University Leipzig) were grown in 75 cm2 flasks (Falcon) at 37°C in a humidified atmosphere at 5% CO2. The medium used was DMEM medium (Gibco BRL) supplemented with 10 % FCS, 1 % penicillin/streptomycin (Gibco BRL), and 0.5 mg/ml fungizone (amphotericin B, Gibco BRL).

Irradiation

EVC 304 cells were trypsinized, counted and 1×105 cells were plated

Ionizing radiation inhibits 20s and 26s proteasome function

There are numerous reports on the use of the ECV 304 cell line as a model for human endothelium. Although this cell line has been recently identified to be a variant of the T24 bladder carcinoma cell line [11], it mimics an endothelial phenotype. In this study, it served as model for cells in an inflammatory environment by exhibiting constitutive and inducible NF-κB and ICAM-1 activity. RAW 264.7 murine macrophages are also a well accepted model for studying inflammatory responses [18].

In order

Discussion

Knowledge about molecular mechanisms activated after application of ionizing radiation is a key to understanding early and late side effects of radiation therapy. Also, identification of the sub-cellular targets of ionizing radiation might uncover new approaches to improve treatment outcome and to minimize toxicity.

Certain aspects of the molecular response to ionizing radiation can appear paradoxical. On one hand, signal transduction pathways leading to production of pro-inflammatory cytokines

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